Atomic Description of Immune Complex that Causes Heparin-Induced Thrombocytopenia

ABSTRACT

The present invention provides a humanized antibody or antibody fragment comprising (a) a humanized light chain comprising 1) Complementarity Determining Region (CDR)-L1, the sequence of which is identical to the sequence of SEQ ID NO: 3; 2) CDR-L2, the sequence of which is identical to the sequence of SEQ ID NO: 4; and 3) CDR-L3, the sequence of which is identical to the sequence of SEQ ID NO: 5, and (b) a humanized heavy chain comprising 1) CDR-H1, the sequence of which is identical to the sequence of SEQ ID NO: 6; 2) CDR-H2, the sequence of which is identical to the sequence of SEQ ID NO: 7; and 3) CDR-H3, the sequence of which is identical to the sequence of SEQ ID NO: 8, as well as methods for treating, diagnosing, and monitoring the progression of HIT. The present invention also provides methods for assessing the antigenicity and ability to cause HIT of anionic anticoagulants. The present invention also provides a mutant protein which has the same amino acid sequence of a wild type PF4 monomer except that (i) at least one amino acid of the wild type PF4 monomer has been deleted, (ii) at least one amino acid of the wild type PF4 monomer has been replaced by another amino acid, or (iii) a combination of such changes has been made. The present invention also provides methods of treating or reducing the likelihood of HIT, treating angiogenesis, treating abnormal cell growth, or affecting coagulation pathologies that lead to thrombus formation, by administering such mutant proteins to a patient.

This application is a continuation of U.S. patent application Ser. No.16/446,902 filed Jun. 20, 2019, which is a continuation of U.S. patentapplication Ser. No. 15/524,511 filed May 4, 2017, which is the NationalStage Application of International Patent Application No.PCT/US2015/059283, filed Nov. 5, 2015, which claims the priority of U.S.Provisional Application No. 62/221,485, filed on Sep. 21, 2015, and U.S.Provisional Application No. 62/076,213, filed Nov. 6, 2014, the contentsof each of which are hereby incorporated by reference.

This invention was made with government support under HL110860 awardedby the National Institutes of Health. The government has certain rightsin the invention.

The instant application contains a Sequence Listing XML, which has beensubmitted electronically as an XML formatted sequence listing with afile name “103241_006875_Sequence_listing.xml” and a creation date ofOct. 27, 2022, and having a size of 26,519 bites. The sequence listingsubmitted electronically is part of the specification and is hereinincorporated by reference in its entirety.

Throughout this application, various publications are referenced,including referenced in parenthesis. Full citations for publicationsreferenced in parenthesis may be found listed at the end of thespecification immediately preceding the claims. The disclosures of allreferenced publications in their entireties are hereby incorporated byreference into this application in order to more fully describe thestate of the art to which this invention pertains.

BACKGROUND OF INVENTION

Heparin is a commonly used anti-coagulant. It prevents the formation ofnew blood clots and stops the enlargement of existing clots. Heparin cansometimes bind to and form a complex with platelet factor 4 (PF4), aprotein made by platelets (cell fragments that aggregate to form bloodclots). Roughly 12 million patients are exposed to heparin annually(Sankar Eke 2014) and up to 1% of these patients will developheparin-induced thrombocytopenia (HIT), a life-threatening complicationwhere patients make antibodies that bind to the heparin/PF4 complex.These antibodies trigger the activation and aggregation of platelets,resulting in blood clots (“thrombosis”) and the depletion of platelets(“thrombocytopenia”).

HIT leads to longer hospital stays and increased morbidity andmortality, resulting in significant additional treatment costs. Forexample, among US medical patients who developed HIT, hospital costswere reported to be higher by an average of —$41,000 compared topatients without HIT (Udeh et al. (2013) Heparin-inducedthrombocytopenia: a clinical and economic review. OA Anaesthetics1(1):3). Healthcare institutions and insurance companies incuradditional costs when HIT is falsely diagnosed, as these misdiagnosedpatients are given unnecessary anti-coagulants (which can be moreexpensive and which can increase the risk of bleeding, leading tosubsequent complications).

Typically management of HIT consists of stopping the heparin and usingan alternative anti-coagulant. These alternative drugs are ofteninhibitors of thrombin, a protein that promotes coagulation. Two directthrombin inhibitors are available in the United States: lepirudin andargatroban.

There is a need for novel compounds, compositions, and methods fordiagnosing, monitoring, and treating HIT.

SUMMARY OF THE INVENTION

The present invention provides a humanized antibody or antibody fragmentcomprising

-   -   (a) a humanized light chain comprising        -   1) Complementarity Determining Region (CDR)-L1, the sequence            of which is identical to the sequence of SEQ ID NO: 3            (KASENVVTYVS);        -   2) CDR-L2, the sequence of which is identical to the            sequence of SEQ ID NO: 4 (GASNRYT); and        -   3) CDR-L3, the sequence of which is identical to the            sequence of SEQ ID NO: 5 (GQGYSYPYT), and    -   (b) a humanized heavy chain comprising        -   1) CDR-H1, the sequence of which is identical to the            sequence of SEQ ID NO: 6 (GFAFSRYDMS);        -   2) CDR-H2, the sequence of which is identical to the            sequence of SEQ ID NO: 7 (TITSGDNYTYYPDSVKG); and        -   3) CDR-H3, the sequence of which is identical to the            sequence of SEQ ID NO: 8 (QGLLYYAMDY).

Aspects of the present invention relate to a vector encoding a humanizedantibody or antibody fragment described herein, or a heavy chain orlight chain of a humanized antibody or antibody fragment of theinvention.

The present invention also provides a cell or virus comprising thevector of the invention, wherein if the cell is a human cell, then thecell is a cultured human cell.

The present invention also provides a method for diagnosingheparin-induced thrombocytopenia (HIT) in a subject, comprising

-   -   i) obtaining a plasma or serum sample from the subject;    -   ii) determining the ratio of        -   (1) the reactivity of antibodies in the sample to wild-type            PF4, to        -   (2) the reactivity of antibodies in the sample to a mutant            PF4 monomer that remains in a monomeric state; and    -   iii) diagnosing the subject as having HIT based on the ratio        of (1) to (2).

The present invention also provides a method for determining whether asubject receiving the administration of heparin or a heparin-mimic is atrisk of becoming afflicted with heparin-induced thrombocytopenia (HIT),comprising

-   -   i) obtaining a plasma or serum sample from the subject;    -   ii) determining the ratio of        -   (1) the reactivity of antibodies in the sample to wild-type            PF4, to        -   (2) the reactivity of antibodies in the sample to a mutant            PF4 monomer that remains in a monomeric state; and    -   iii) identifying the subject as at risk of becoming afflicted        with HIT based on the ratio of (1) to (2).

The present invention also provides a method for monitoring theprogression of heparin-induced thrombocytopenia (HIT) in a subject,comprising

-   -   i) obtaining a first plasma or serum sample from the subject;    -   ii) obtaining a second plasma or serum sample from the subject;    -   iii) determining in each of the first and second samples of the        subject the ratio of        -   (1) the reactivity of antibodies in the sample to wild-type            PF4, to        -   (2) the reactivity of antibodies in the sample to a mutant            PF4 monomer that remains in a monomeric state; and    -   iv) determining that HIT has progressed in the subject if the        ratio of (1) to (2) is higher in the second sample than in the        first sample, and determining that the HIT has not progressed in        the subject if the ratio of (1) to (2) is lower in the second        sample than in the first sample or if the ratio of (1) to (2) is        the same in the first and second samples.

Aspects of the present invention relate to a method for treating asubject afflicted with heparin-induced thrombocytopenia (HIT) comprisingadministering to the subject an effective amount of an antibody orantibody fragment that is capable of binding at least a portion of thesame epitope as the epitope to which RTO binds so as to reduce plateletfactor 4 (PF4) oligomerization.

The present invention also provides a method for reducing the likelihoodthat a subject receiving the administration of heparin or aheparin-mimic will become afflicted with heparin-inducedthrombocytopenia (HIT) comprising administering to the subject aneffective amount of an antibody or antibody fragment that is capable ofbinding at least a portion of the same epitope as the epitope to whichRTO binds so as to reduce platelet factor 4 (PF4) oligomerization.

The present invention also provides a method for assessing theantigenicity of an anionic anticoagulant comprising

-   -   i) obtaining a plasma or serum sample from a subject who has        received administration of the anionic anticoagulant;    -   ii) determining the ratio of        -   (1) the reactivity of antibodies in the sample to wild-type            PF4, to        -   (2) the reactivity of antibodies in the sample to a mutant            PF4 monomer that remains in a monomeric state; and    -   iii) assessing the antigenicity of the anionic anticoagulant        based on the ratio of (1) to (2).

Aspects of the present invention relate to a method for assessingwhether an anionic anticoagulant is capable of causing heparin-inducedthrombocytopenia (HIT) comprising

-   -   i) obtaining a plasma or serum sample from a subject who has        received administration of the anionic anticoagulant;    -   ii) determining the ratio of        -   (1) the reactivity of antibodies in the sample to wild-type            PF4, to        -   (2) the reactivity of antibodies in the sample to a mutant            PF4 monomer that remains in a monomeric state; and    -   iii) assessing whether the anionic anticoagulant is capable of        causing HIT based on the ratio of (1) to (2).

The present invention also provides a mutant platelet factor 4 (PF4)monomer which has the same amino acid sequence of a wild type PF4monomer except that

-   -   (i) at least one amino acid of the wild type PF4 monomer has        been deleted,    -   (ii) at least one amino acid of the wild type PF4 monomer has        been replaced by another amino acid, or    -   (iii) a combination of such changes has been made.

The present invention also provides a method for treating a subjectafflicted with heparin-induced thrombocytopenia (HIT), treatingangiogenesis, treating abnormal cell growth, or affecting coagulationpathologies that lead to thrombus formation, comprising administering tothe subject an effective amount of a mutant platelet factor 4 (PF4)monomer so as to interfere with PF4 tetramer oligomerization.

The present invention also provides a method for reducing the likelihoodthat a subject receiving the administration of heparin or aheparin-mimic will become afflicted with heparin-inducedthrombocytopenia (HIT) comprising administering to the subject aneffective amount of a mutant platelet factor 4 (PF4) monomer so as tointerfere with PF4 tetramer oligomerization.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A and FIG. 1B. Well-defined fondaparinux electron density.

FIG. 1A—The 2Fo-Fc electron density map (at 1.4a contour level) fittedwith a fondaparinux in the PF4/fondaparinux complex. FIG. 1B—the 2Fo-FcOMIT electron density in the fondaparinux binding site, contoured at 1.4σ. The positions of the ordered sulfate groups are confirmed by theanomalous signals of sulfur.

FIG. 2 . The asymmetric ‘open’-‘close’ conformation in thePF4/fondaparinux complex crystal. Measurement of the E28 to K50distances shows that PF4 in the PF4/fondaparinux complex crystal adoptsan asymmetric ‘open’-‘closed’ conformation, which is almost same as inthe apo-PF4 crystal structure. Salt bridges exist between chain A andchain C, not between chain B and chain D, in the asymetric tetramer.Fondaparinux binds only to the close end of PF4 tetramer and stabilizesthe tetramer structure. Only two binding grooves for fondaparinux arepresent on the surface of the PF4 tetramer due to the asymmetry.

FIG. 3 . Structure-based mutations of KKO epitopes on the PF4 surface.Sequence alignment of human PF4 (SEQ ID NO: 11) with mouse PF4 (SEQ IDNO: 12) guided inventors to make structure-based mutations in the KKOepitope (FIG. 2B). PF4-SCV⁹⁻¹¹; PF4-R⁵⁵.

FIG. 4 . KKO recognize the surface of the ‘open’ end on the PF4 in theKKOFab/PF4 complex crystal. Measurement of the E28 to K50 distances(salt bridges exist between chain A and chain C, not between chain B andchain D, in the asymetric tetramer, FIG. 2 ) reveals that PF4 in theKKOFab/PF4 complex adopts a symmetric ‘open’-‘open’ conformation. Thisfinding provides evidence that the HIT antibody KKO recognizes themolecular surface of the ‘open’ end on a PF4 tetramer, which is exposedafter the stabilization of the tetramer structure by a heparin.

FIG. 5 . FPLC profiles of the RTO-Fab/PF4 Complex. Gel filtrationprofiles of wild type PF4 only, RTOFab only and RTOFab/PF4 complex on aSuperdex 75 (GE Healthcare) column. The Superdex 75 column wascalibrated using standard proteins and then molecular weight of eachpeak was determined. The difference between the molecular weight of theRTOFab/PF4 complex peak and that of the RTOFab only peak is about 7,000dalton, which corresponds to the molecular weight of a PF4 monomer.

FIG. 6 . Comparison of the RTO epitope on a PF4 monomer and the KKOepitope on a PF4 tetramer. Arrows on the bottom denote overlapping sitesof interactions on PF4. The RTO epitope on PF4 (SEQ ID NO: 10) overlapswith the KKO epitope (SEQ ID NO: 9), especially within the A32-A39 loop.

FIG. 7A and FIG. 7B. RTO Sequences and Epitopes. FIG. 7A—RTO Sequences(Light Chain RTO-Fab, SEQ ID NO: 1; Heavy Chain RTO-Fab, SEQ ID NO: 2;CDR-L1, SEQ ID NO: 3; CDR-L2, SEQ ID NO: 4; CDR-L3, SEQ ID NO: 5; SEQ IDNO: 6; CDR-H2, SEQ ID NO: 7; CDR-H3, SEQ ID NO: 8). FIG. 7B RTO bindingto PF4 (SEQ ID NO: 10).

FIG. 8 . Humanized RTO (huRTO) sequences. huRTO-L (SEQ ID NO: 13) andhuRTO-H (SEQ ID NO: 14).

FIG. 9A, FIG. 9B, FIG. 9C, FIG. 9D, FIG. 9E, and FIG. 9F. Crystalstructure of the PF4/fondaparinux complex and formation of the HITantigen. FIG. 9A—Overall structure of the PF4/pentasaccharide complex.Fondaparinux makes contacts with a single PF4 tetramer in the grooveamong the monomers on one side of the asymmetric tetramer. Monomers A,B, C and D in one PF4 tetramer are indicated. FIG. 9B—Fondaparinux(stick representations) stabilizes the PF4 tetramer by binding in thegroove among 3 monomers in a PF4 tetramer. Dotted line indicates thepolar interactions between Fondaparinux and three PF4 monomers. FIG.9C—One fondaparinux (spheres) denoted in the smaller box binds in thegroove of one tetramer (cartoon representation on the left), and alsobinds to the C-terminal helix of a second tetramer (cartoonrepresentation on the right), thereby bridging PF4 tetramers. FIG.9D—Electrostatic potential surface representation of the PF4 tetramershows that fondaparinux binds along a continuous positively chargedsurface on the “closed” side of PF4 tetramer. FIG. 9E—Detailedrepresentation of the positively charged residues (labeled) on thefondaparinux binding interface between two PF4 tetramers. FIG.9F—Analysis of crystal lattice reveals a molecular pathway of theformation of antigenic complexes. A fragment of heparin first bindswithin the groove of one PF4 tetramer (left); binding of the first PF4tetramer imparts a local linearized structure on heparin, which enhancesthe binding of a second tetramer (middle); progression of this processeventuates in the formation of ultralarge antigenic complexes (right).

FIG. 10A, FIG. 10B, FIG. 10C, FIG. 10D, and FIG. 10E. Crystal structureof PF4/KKO-Fab complex. FIG. 10A—Overall structure of the PF4/KKO-Fabcomplex (upper panel: cartoon representations of the complex; lowerpanel: molecular surface representations). The heavy chain and lightchain of KKO-Fab are depicted. FIG. 10B—Detailed binding interface ofHIT antibody KKO to a PF4 tetramer. Residues within a PF4 tetramer thatare less than 5 Å away from KKO-Fab molecule are underlined. PF4monomers are labeled (i.e. Chain A, B, C, D) as in FIG. 9A. The sequenceshown is SEQ ID NO: 9. FIG. 10C—Binding of KKO and RTO tostructure-based PF4 mutants. FIG. 10D—Platelet aggregation by wild-typePF4 and PF4 mutants. KKO induced platelet aggregation in the presence ofwild-type PF4 and heparin whereas an isotype matched non-pathogenicantibody RTO described below did not. The panel also demonstrates thatPF4 mutants bearing mutations along the KKO binding interface wereunable to mediate KKO-induced platelet aggregation. FIG. 10E—Model ofthe KKO-Fab/PF4/heparin ternary complex. Surface representations ofKKO-Fab are depicted. The model assumes the heparin molecule is composedof about 7 structures similar to fondaparinux depicted in the figure asa non-continuous chain. Intact UFH may further enhance the stability ofthe holo complex compared with the fondaparinux fragment, therebyrendering it more antigenic and more capable of binding multiple IgGantibodies.

FIG. 11A, FIG. 11B, FIG. 11C, and FIG. 11D. Crystal structure ofPF4/RTO-Fab. FIG. 11A—Overall structure of the PF4/RTO-Fab complex (leftpanel: cartoon representations of the complex; right panel: molecularsurface representations). The heavy chain and light chain of RTO-Fab aredepicted. FIG. 11B—Detailed binding interface of the non-HIT antibodyRTO to a PF4 monomer. Residues of a PF4 monomer that are less than 5 Åaway from RTO-Fab molecule are underlined. The sequence shown is SEQ IDNO: 10. FIG. 11C—Superposition of the PF4 monomer (on the left) in theRTO-Fab/PF4 complex with that in the unbound PF4 (on the right)indicates that binding of RTO-Fab causes a dramatic structural change inthe PF4 monomer: the C-terminal helices are shifted ˜60°. FIG.11D—Superposition of the PF4 monomer (middle) in complex with RTO-Fab(labeled) with the unbound PF4 tetramer (right). The three arrows on theleft of the diagram indicate the sites where binding of RTOFab (left) toone PF4 monomer causes steric clashes with a second PF4 monomer in thetetramer, thereby preventing tetramer formation.

FIG. 12A, FIG. 12B, FIG. 12C, and FIG. 12D. RTO prevents KKO-inducedplatelet activation and platelet aggregation in vitro and thrombosis invivo. FIG. 12A—Inhibition of KKO/PF4 mediated platelet activation byRTO. Samples of whole blood were incubated with the indicatedconcentrations of RTO in the presence of PF4 (10 μg/ml) for 15 minbefore adding the platelet-activating anti-PF4 antibody KKO or humanIgG. Activation of platelets was followed by expression of P-selectin;the effect of RTO is expressed as % of geometric mean fluorescentintensity (MFI) of P-selectin expression on platelets relative to MFI inthe absence of RTO. FIG. 12B—In vitro platelet activation assaydemonstrated that preincubation of PF4 with RTO prevented KKO-inducedplatelet aggregation. FIG. 12C—Representative composite images ofplatelet fluorescence overlaid on brightfield snapshots of injuries inmice receiving either RTO or the IgGk2B isotype control TRA are shown.Pre KKO images show thrombi 15 minutes after initial injury andinjection of RTO or TRA. Post KKO images represent the same thrombus 15minutes after KKO had been injected intravenously. Arrows represent thedirection of blood flow. FIG. 12D—Each dot denotes the percent change inthe size of a single injury based on binding of fluorescently labeledplatelets in mice receiving either RTO or the IgGK2B isotype control TRAfollowed by KKO. Error bars show the standard deviation. N=18 injuriesin 3 mice for RTO, N=19 injuries in 3 mice for TRA. p<0.0001.

FIG. 13 . A cartoon summarizing a model of the pathogenesis ofheparin-induced thrombocytopenia and the use of RTO as an inhibitor. PF4molecules (larger circles) exist in an equilibrium among monomers,dimers and tetramers. Upon binding to heparin (smaller circles), theconfiguration of the tetramer is stabilized. As a result, the open endof the PF4 tetramer is oriented and recognized by HIT-antibody KKO(black). KKO in turn fosters PF4 oligomerization and workscollaboratively with heparin to stabilize the ternary complex. The netresult is the generation of stable ultralarge immune complexes capableof sustained activation of Fcγ receptors on platelets and monocyteswhich consequently leads to HIT. The non-HIT isotype-matched antibodyRTO (labelled, lighter gray) binds to PF4 monomers, prevents PF4oligomerization, prevents formation of ultralarge immune complexes andas a result may prevent HIT. The cartoon assumes heparin is composed ofabout 7 structures similar to fondaparinux.

FIG. 14A and FIG. 14B. FIG. 14A—The protein sequence alignment of wildtype PF4 (SEQ ID NO: 11) and Del 8AA mutant (SEQ ID NO: 16). FIG.14B—structures of wild type PF4 (PDB id:1RHP, as tetramer) and Del 8AAmutant (modeled from RTOFab/PF4 monomer complex, PDB code 4RAU, asmonomer) are shown.

FIG. 15 . FPLC profiles of wild type PF4 in DPBS, PBS/0.5M NaCl, PF4mutant V29W, and PF4 mutant Del8AA-K50E are shown. Aconcentration-dependent monomer-dimertetramer equilibrium can beobserved for wild type PF4 in DPBS. The retention time of PF4 mutantV29W corresponds to a dimer/monomer mixture. And the peak for PF4 mutantDel8AA-K50E demonstrates that the mutant Del8AA-K50E mainly exists asmonomer in DPBS.

FIG. 16 and FIG. 17 . PF4 and PF4 mutants were expressed in S2 insectcells and purified by heparin affinity column. The mixtures of wt PF4and Delta 8AA PF4 were at 5 ug/ml total, with percentage of Delta 8AAPF4 in the mixture increasing from 0 to 100%. The wt PF4 and Delta 8AAPF4 were pre-incubated together for 30 min and then heparin at 0.1 u/ml(FIG. 16 ) or no heparin (FIG. 17 ) was added to the mixture. Themixtures were loaded onto ELISA plate wells overnight. The next day, KKOELISA was performed. Data shows even 5% of Delta 8AA PF4 in the mixtureis sufficient to inhibit KKO binding essentially.

FIG. 18A and FIG. 18B. Images of a portion of the 2Fo-Fc electrondensity map (at 1.5σ contour level) for the KKO-Fab/PF4 tetramer complex(FIG. 18A) and the 2Fo-Fc electron density map (at 1.3σ contour level)for the RTO-Fab/PF4 monomer complex (FIG. 18B).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a humanized antibody or antibody fragmentcomprising

-   -   (a) a humanized light chain comprising        -   1) Complementarity Determining Region (CDR)-L1, the sequence            of which is identical to the sequence of SEQ ID NO: 3            (KASENVVTYVS);        -   2) CDR-L2, the sequence of which is identical to the            sequence of SEQ ID NO: 4 (GASNRYT); and        -   3) CDR-L3, the sequence of which is identical to the            sequence of SEQ ID NO: 5 (GQGYSYPYT), and    -   (b) a humanized heavy chain comprising        -   1) CDR-H1, the sequence of which is identical to the            sequence of SEQ ID NO: 6 (GFAFSRYDMS);        -   2) CDR-H2, the sequence of which is identical to the            sequence of SEQ ID NO: 7 (TITSGDNYTYYPDSVKG); and        -   3) CDR-H3, the sequence of which is identical to the            sequence of SEQ ID NO: 8 (QGLLYYAMDY).

In some embodiments, the humanized antibody or antibody fragment iscapable of binding at least a portion of the same epitope as the epitopeto which RTO binds so as to reduce platelet factor 4 (PF4)oligomerization.

In some embodiments, the humanized antibody or antibody fragment iscapable of binding to a portion of the same epitope as the epitope towhich RTO binds that overlaps with a portion of the same epitope as theepitope to which KKO binds.

In some embodiments, the humanized antibody or antibody fragment iscapable of binding to the same epitope as the epitope to which RTObinds.

In some embodiments, the humanized antibody or antibody fragment iscapable of reducing PF4 oligomerization.

In some embodiments, the oligomerization is dimerization ortetramerization.

In some embodiments, the humanized antibody or antibody fragment iscapable of reducing the binding of KKO to PF4.

In some embodiments, the humanized antibody or antibody fragment iscapable of binding between the PF4 AB dimer interface.

In some embodiments, the humanized antibody comprises a human IgG1,IgG2, IgG3, IgG4, IgM, IgE, or IgA heavy chain immunoglobulin constantdomain.

In some embodiments, the fragment is a Fv, Fab, Fab′, Fab′-SH, F(ab′)₂or a single-chain antibody molecule, or part of a diabody or amultispecific antibody formed from more than one antibody fragment.

In some embodiments, the humanized antibody or antibody fragmentcomprises

-   -   (a) a humanized light chain having amino acids in the sequence        of SEQ ID NO: 13; and    -   (b) a humanized heavy chain having amino acids in the sequence        of SEQ ID NO: 14.

In some embodiments, the humanized antibody or antibody fragment is anisolated humanized antibody or antibody fragment.

The present invention also provides a pharmaceutical compositioncomprising a humanized antibody or antibody fragment of the invention.

Aspects of the present invention relate to a vector encoding a humanizedantibody or antibody fragment described herein, or a heavy chain orlight chain of a humanized antibody or antibody fragment of theinvention.

The present invention also provides a cell or virus comprising thevector of the invention, wherein if the cell is a human cell, then thecell is a cultured human cell.

The present invention also provides a method for diagnosingheparin-induced thrombocytopenia (HIT) in a subject, comprising

-   -   i) obtaining a plasma or serum sample from the subject;    -   ii) determining the ratio of        -   (1) the reactivity of antibodies in the sample to wild-type            PF4, to        -   (2) the reactivity of antibodies in the sample to a mutant            PF4 monomer that remains in a monomeric state; and    -   iii) diagnosing the subject as having HIT based on the ratio        of (1) to (2).

In some embodiments, the subject is diagnosed as having HIT if the ratioof (1) to (2) in the sample is equal to or greater than a referenceratio of (1) to (2) obtained from a plasma or serum sample from asubject or group of subjects afflicted with HIT.

The present invention also provides a method for determining whether asubject receiving the administration of heparin or a heparin-mimic is atrisk of becoming afflicted with heparin-induced thrombocytopenia (HIT),comprising

-   -   i) obtaining a plasma or serum sample from the subject;    -   ii) determining the ratio of        -   (1) the reactivity of antibodies in the sample to wild-type            PF4, to        -   (2) the reactivity of antibodies in the sample to a mutant            PF4 monomer that remains in a monomeric state; and    -   iii) identifying the subject as at risk of becoming afflicted        with HIT based on the ratio of (1) to (2).

In some embodiments, the subject is identified as at risk of becomingafflicted with HIT if the ratio of (1) to (2) in the sample is equal toor greater than a reference ratio of (1) to (2) obtained from a plasmaor serum sample from a subject or group of subjects not afflicted withHIT, or if the ratio of (1) to (2) in the sample is equal to or greaterthan a reference ratio of (1) to (2) obtained from a serum or plasmasample taken from the subject before the heparin or the heparin-mimicwas administered to the subject.

The present invention also provides a method for monitoring theprogression of heparin-induced thrombocytopenia (HIT) in a subject,comprising

-   -   i) obtaining a first plasma or serum sample from the subject;    -   ii) obtaining a second plasma or serum sample from the subject;    -   iii) determining in each of the first and second samples of the        subject the ratio of        -   (1) the reactivity of antibodies in the sample to wild-type            PF4, to        -   (2) the reactivity of antibodies in the sample to a mutant            PF4 monomer that remains in a monomeric state; and    -   iv) determining that HIT has progressed in the subject if the        ratio of (1) to (2) is higher in the second sample than in the        first sample, and determining that the HIT has not progressed in        the subject if the ratio of (1) to (2) is lower in the second        sample than in the first sample or if the ratio of (1) to (2) is        the same in the first and second samples.

In some embodiments, determining the ratio of (1) and (2) comprisesELISA.

In some embodiments, the ratio of (1) to (2) is obtained using theoptical density for (1) and the optical density for (2) obtained usingELISA.

Aspects of the present invention relate to a method for treating asubject afflicted with heparin-induced thrombocytopenia (HIT) comprisingadministering to the subject an effective amount of an antibody orantibody fragment that is capable of binding at least a portion of thesame epitope as the epitope to which RTO binds so as to reduce plateletfactor 4 (PF4) oligomerization.

The present invention also provides a method for reducing the likelihoodthat a subject receiving the administration of heparin or aheparin-mimic will become afflicted with heparin-inducedthrombocytopenia (HIT) comprising administering to the subject aneffective amount of an antibody or antibody fragment that is capable ofbinding at least a portion of the same epitope as the epitope to whichRTO binds so as to reduce platelet factor 4 (PF4) oligomerization.

In some embodiments, the antibody or antibody fragment is

-   -   (a) RTO or a fragment of RTO;    -   (b) a humanized antibody or antibody fragment;    -   (c) capable of binding to the same epitope as the epitope to        which RTO binds;    -   (d) capable of binding to a portion of the same epitope as the        epitope to which RTO binds that overlaps with a portion of the        same epitope as the epitope to which KKO binds;    -   (e) capable of reducing the binding of KKO to PF4; or capable of        binding between the PF4 AB dimer interface.

In some embodiments, heparin is being or has been administered to thesubject.

In some embodiments, the heparin is low-molecular-weight heparin orunfractionated heparin.

In some embodiments, a heparin mimic is being or has been administeredto the subject.

In some embodiments, the heparin mimic is fondaparinux.

In some embodiments, HIT has been diagnosed in the subject according toa method of the present invention.

In some embodiments, the subject has been identified as at risk ofbecoming afflicted with HIT according to a method of the presentinvention.

In some embodiments, the progression of HIT has been monitored in thesubject according to a method of the present invention.

In some embodiments, a method of treating a subject comprises monitoringthe progression of HIT in the subject according to a method of thepresent invention.

The present invention also provides a method for assessing theantigenicity of an anionic anticoagulant comprising

-   -   i) obtaining a plasma or serum sample from a subject who has        received administration of the anionic anticoagulant;    -   ii) determining the ratio of        -   (1) the reactivity of antibodies in the sample to wild-type            PF4, to        -   (2) the reactivity of antibodies in the sample to a mutant            PF4 monomer that remains in a monomeric state; and    -   iii) assessing the antigenicity of the anionic anticoagulant        based on the ratio of (1) to (2).

In some embodiments, the anionic anticoagulant is identified to becapable of increasing the production of pathogenic antibodies insubjects if the ratio of (1) to (2) in the sample is equal to or greaterthan a reference ratio of (1) to (2) obtained from a plasma or serumsample from a subject or group of subjects not afflicted with HIT, or ifthe ratio of (1) to (2) in the sample is equal to or greater than areference ratio of (1) to (2) obtained from a serum or plasma sampletaken from the subject before the heparin or the heparin-mimic wasadministered to the subject.

Aspects of the present invention relate to a method for assessingwhether an anionic anticoagulant is capable of causing heparin-inducedthrombocytopenia (HIT) comprising

-   -   i) obtaining a plasma or serum sample from a subject who has        received administration of the anionic anticoagulant;    -   ii) determining the ratio of        -   (1) the reactivity of antibodies in the sample to wild-type            PF4, to        -   (2) the reactivity of antibodies in the sample to a mutant            PF4 monomer that remains in a monomeric state; and    -   iii) assessing whether the anionic anticoagulant is capable of        causing HIT based on the ratio of (1) to (2).

In some embodiments, the anionic anticoagulant is identified to becapable of causing HIT if the ratio of (1) to (2) in the sample is equalto or greater than a reference ratio of (1) to (2) obtained from aplasma or serum sample from a subject or group of subjects not afflictedwith HIT, or if the ratio of (1) to (2) in the sample is equal to orgreater than a reference ratio of (1) to (2) obtained from a serum orplasma sample taken from the subject before the heparin or theheparin-mimic was administered to the subject.

In some embodiments, determining the ratio of (1) and (2) comprisesELISA.

In some embodiments, the ratio of (1) to (2) is obtained using theoptical density for (1) and the optical density for (2) obtained usingELISA.

In some embodiments, the subject is a mammalian subject.

In some embodiments, the subject is a human subject.

The present invention provides a mutant protein which has the same aminoacid sequence of a wild type PF4 monomer except that

-   -   (i) at least one amino acid of the wild type PF4 monomer has        been deleted,    -   (ii) at least one amino acid of the wild type PF4 monomer has        been replaced by another amino acid, or    -   (iii) a combination of such changes has been made.

In some embodiments, the mutant protein the at least one amino acid thathas been deleted or replaced is on the N-terminal end of the mutant PF4monomer.

In some embodiments, at least 8 amino acids are deleted from theN-terminal end of a wild type PF4 monomer.

In one embodiment, the mutant PF4 monomer is a K53E mutant.

In some embodiments, the amino acid sequence of the mutant protein isset forth is SEQ ID NO: 15.

In some embodiments, the mutant protein interferes with wild type PF4tetramer oligomerization.

The present invention also provides a method for treating a subjectafflicted with heparin-induced thrombocytopenia (HIT) comprisingadministering to the subject an effective amount of a mutant PF4 monomerso as to interfere with PF4 tetramer oligomerization.

The present invention also provides a method for reducing the likelihoodthat a subject receiving the administration of heparin or aheparin-mimic will become afflicted with heparin-inducedthrombocytopenia (HIT) comprising administering to the subject aneffective amount of a mutant PF4 monomer so as to interfere with PF4tetramer oligomerization.

The present invention also provides a method for treating coagulationpathologies that lead to thrombus formation, angiogenesis or abnormalcell growth comprising administering to the subject an effective amountof a mutant platelet factor 4 (PF4) monomer so as to interfere with PF4tetramer oligomerization.

Each embodiment disclosed herein is contemplated as being applicable toeach of the other disclosed embodiments. Thus, all combinations of thevarious elements described herein are within the scope of the invention.

Diagnostic and Screening Assays

Patients have pathogenic and non-pathogenic anti-PF4 antibodies in theirplasmas. The proportion of each type of antibodies varies among patientsbut this difference cannot be detected using current ELISAs because thewells contain PF4 monomers, tetramers and various sized oligomers.

Identifying antibodies that bind to monomers, i.e. are nonpathogenic,helps to eliminate the detection of clinically irrelevant antibodiesthat current cause false positive diagnoses, label patients as allergicto heparin and cause them to receive expensive anticoagulants thatcannot be reversed if bleeding occurs. Therefore there is a need todevelop a new assay that provides a higher true positive result ratethan is currently available.

The logic for diagnostic assays provided in the present invention is asfollows.

1. Oligomerization of PF4 is a fundamental step in the pathogenesis ofHIT2. PF4 is synthesized as a monomer but oligomerizes at highconcentrations, a process that is enhanced to a variable extent byanionic polysaccharides, some of which are used as anticoagulants, e.g.heparins3. KKO identifies the oligomeric (pathogenic) state of PF44. A mutant PF4 monomer that does not oligomerize (e.g. by deletion of 8amino acids from the N-terminal) prevents the formation of a tetramerwith a KKO binding site, therefore KKO binds to wild type PF4 but not tothe mutant monomer;5. RTO or other non-pathogenic antibodies bind to monomeric PF4 andmonomeric wild type (wt) PF4 to the same or similar extent

Based on this the present invention provides a test in which one targetis wt PF4 and one target is a mutant PF4 monomer that remains in themonomeric state. Pathogenic antibodies in plasma will bind to wt PF4 butnot the mutant PF4 monomer. In contrast, non-pathogenic antibodies willbind to wt PF4 and the mutant PF4 monomer. Therefore the OD wtPF4/OD M11can be used to distinguish patients at high risk of HIT from the fargreater number of individuals who develop clinically irrelevantantibodies that now give false positive results.

Mutant PF4 monomers that remain in the monomeric state can also be usedfor this purpose.

Another variation on this concept is that the increase in the ODwtPF4/OD mutant PF4 monomer ratio can be used to assess the antigenicityof existing and potential new anionic anticoagulants.

WT-PF4: sequence (SEQ ID NO: 11) EAEEDGDLQCLCVKTTSQVRPRHITSLEVIKAGPHCPTAQLIATLKNGRKICLDLQAPLYKKIIKKLLES

Terms

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by a person of ordinaryskill in the art to which this invention belongs.

As used herein, and unless stated otherwise or required otherwise bycontext, each of the following terms shall have the definition set forthbelow.

As used herein, “about” in the context of a numerical value or rangemeans±10% of the numerical value or range recited or claimed, unless thecontext requires a more limited range.

It is understood that where a parameter range is provided, all integerswithin that range, and tenths thereof, are also provided by theinvention. For example, “0.2-5 mg/kg/day” is a disclosure of 0.2mg/kg/day, 0.3 mg/kg/day, 0.4 mg/kg/day, 0.5 mg/kg/day, 0.6 mg/kg/dayetc. up to 5.0 mg/kg/day.

The terms “treating” or “treatment” refer to any success or indicia ofsuccess in the attenuation or amelioration of an injury, pathology orcondition, including any objective or subjective parameter such asabatement, remission, diminishing of symptoms or making the injury,pathology, or condition more tolerable to the patient, slowing in therate of degeneration or decline, making the final point of degenerationless debilitating, improving a subject's physical or mental well-being,or prolonging the length of survival. The treatment or amelioration ofsymptoms can be based on objective or subjective parameters; includingthe results of a physical examination, neurological examination, and/orpsychiatric evaluations.

“Effective amount” and “therapeutically effective amount” are usedinterchangeably herein, and refer to an amount of a compound effectiveto achieve a particular biological or therapeutic result such as, butnot limited to, biological or therapeutic results disclosed, described,or exemplified herein. A therapeutically effective amount of thecompound may vary according to factors such as the disease state, age,sex, and weight of the subject, and the ability of the antibody orantigen-binding fragment thereof to elicit a desired response in thesubject. In embodiments of the invention, such results may include, butare not limited to, the treatment of heparin-induced thrombocytopenia(HIT), as determined by any means suitable in the art.

Antibodies

The term “antibody” is used in the broadest sense and specificallycovers monoclonal antibodies (including full length monoclonalantibodies), polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), monovalent antibodies, and multivalentantibodies. Additionally, the term “antibody” refers to all isotypes ofimmunoglobulins (IgG, IgA, IgE, IgM, IgD, and IgY) including variousmonomeric and polymeric forms of each isotype, unless otherwisespecified.

“Antibody fragments” comprise a portion of a full length antibody,generally the antigen binding or variable region thereof. Examples ofantibody fragments include but are not limited to Fv, Fab, Fab′,Fab′-SH, F(ab′)2; diabodies; linear antibodies; single-chain antibodymolecules (e.g., scFv); and multispecific antibodies formed fromantibody fragments. Various techniques have been developed for theproduction of antibody fragments, including proteolytic digestion ofantibodies and recombinant production in host cells; however, othertechniques for the production of antibody fragments will be apparent tothe skilled practitioner. In some embodiments, the antibody fragment ofchoice is a single chain Fv fragment (scFv). “Single-chain Fv” or “scFv”antibody fragments comprise the V H and V L domains of antibody, whereinthese domains are present in a single polypeptide chain. Generally, theFv polypeptide further comprises a polypeptide linker between the V Hand V L domains which enables the scFv to form the desired structure forantigen binding. For a review of scFv and other antibody fragments, seeJames D. Marks, Antibody Engineering, Chapter 2, Oxford University Press(1995) (Carl K. Borrebaeck, Ed.).

The term “epitope” refers to a portion of a molecule (the antigen) thatis capable of being bound by a binding agent, e.g., an antibody, at oneor more of the binding agent's antigen binding regions. Epitopes usuallyconsist of specific three-dimensional structural characteristics, aswell as specific charge characteristics.

As used herein, “monoclonal antibody” means an antibody obtained from apopulation of substantially homogeneous antibodies, i.e., the individualantibodies comprising the population are identical except for possiblenaturally occurring mutations that may be present in minor amounts.Monoclonal antibodies are highly specific, being directed against asingle antigenic site. Furthermore, in contrast to conventional(polyclonal) antibody preparations that typically include differentantibodies directed against different determinants, each monoclonalantibody is directed against a single determinant on the antigen. Themodifier “monoclonal” indicates the character of the antibody as beingobtained from a substantially homogeneous population of antibodies, andis not to be construed as requiring production of the antibody by anyparticular method. For example, the monoclonal antibodies to be used inaccordance with the present invention may be made by the hybridomamethod first described by Kohler and Milstein, Nature 256:495-97 (1975),or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No.4,816,567). The monoclonal antibodies may also be isolated from phagedisplay libraries using the techniques described, for example, inClackson et al., Nature 352:624-28 (1991) and Marks et al., J. Mol.Biol. 222(3):581-97 (1991).

The term “hybridoma” or “hybridoma cell line” refers to a cell linederived by cell fusion, or somatic cell hybridization, between a normallymphocyte and an immortalized lymphocyte tumor line. In particular, Bcell hybridomas are created by fusion of normal B cells of definedantigen specificity with a myeloma cell line, to yield immortal celllines that produce monoclonal antibodies. In general, techniques forproducing human B cell hybridomas, are well known in the art (Kozbor etal., Immunol. Today 4:72 (1983); Cole et al., in Monoclonal Antibodiesand Cancer Therapy, Alan R. Liss, Inc. 77-96 (1985)).

The term “humanized antibodies” means antibodies that contain minimalsequence derived from non-human immunoglobulin sequences. For the mostpart, humanized antibodies are human immunoglobulins (recipientantibody) in which residues from a hyper variable region of therecipient are replaced by residues from a hypervariable region of anon-human species (donor antibody) such as mouse, rat, rabbit ornonhuman primate having the desired specificity, affinity, and capacity.See, for example, U.S. Pat. Nos. 5,225,539; 5,585,089; 5,693,761;5,693,762; 5,859,205, each herein incorporated by reference. In someinstances, framework residues of the human immunoglobulin are replacedby corresponding non-human residues (see, for example, U.S. Pat. Nos.5,585,089; 5,693,761; 5,693,762, each herein incorporated by reference).Furthermore, humanized antibodies may comprise residues that are notfound in the recipient antibody or in the donor antibody. Thesemodifications are made to further refine antibody performance (e.g., toobtain desired affinity). In general, the humanized antibody willcomprise substantially all of at least one, and typically two, variabledomains, in which all or substantially all of the hypervariable regionscorrespond to those of a non-human immunoglobulin and all orsubstantially all of the framework regions are those of a humanimmunoglobulin sequence. The humanized antibody optionally also willcomprise at least a portion of an immunoglobulin constant region (Fc),typically that of a human immunoglobulin. For further details see Joneset al., Nature 331:522-25 (1986); Riechmann et al., Nature 332:323-27(1988); and Presta, Curro Opin. Struct. Biol. 2:593-96 (1992), each ofwhich is incorporated herein by reference.

An immunoglobulin light or heavy chain variable region consists of a“framework” region interrupted by three hypervariable regions. Thus, theterm “hypervariable region” refers to the amino acid residues of anantibody which are responsible for antigen binding. The hypervariableregion comprises amino acid residues from a “Complementarity DeterminingRegion” or “CDR” and/or those residues from a “hypervariable loop” inthe heavy chain variable domain; Chothia and Lesk, 1987, J. Mol. Biol.196: 901-917). “Framework Region” or “FR” residues are those variabledomain residues other than the hypervariable region residues as hereindefined. The sequences of the framework regions of different light orheavy chains are relatively conserved within a species. Thus, a “humanframework region” is a framework region that is substantially identical(about 85% or more, usually 90-95% or more, such as 100%) to theframework region of a naturally occurring human immunoglobulin. Theframework region of an antibody, that is the combined framework regionsof the constituent light and heavy chains, serves to position and alignthe CDR's. The CDR's are primarily responsible for binding to an epitopeof an antigen. Antibodies of the invention also include antibodiesproduced in a non-human mammalian host, more particularly a transgenicmouse, characterized by inactivated endogenous immunoglobulin (Ig) loci.In such transgenic animals, competent endogenous genes for theexpression of light and heavy subunits of host immunoglobulins arerendered non-functional and substituted with the analogous humanimmunoglobulin loci. These transgenic animals produce human antibodiesin the substantial absence of light or heavy host immunoglobulinsubunits. See, for example, U.S. Pat. No. 5,939,598, the entire contentsof which are incorporated herein by reference.

Those skilled in the art will be aware of how to produce antibodymolecules of the present invention. For example, polyclonal antisera ormonoclonal antibodies can be made using standard methods. A mammal,(e.g., a mouse, hamster, or rabbit) can be immunized with an immunogenicform of the protein which elicits an antibody response in the mammal.For instance, a mammal can be immunized with irradiated cells that weretransfected with a nucleic acid encoding the protein such that highlevels of the protein were expressed on the cell surface. The progressof immunization can be monitored by detection of antibody titers inplasma or serum. Standard ELISA or other immunoassay can be used withthe immunogen as antigen to assess the levels of antibodies. Followingimmunization, anti sera can be obtained, and, if desired IgG moleculescorresponding to the polyclonal antibodies may be isolated from thesera.

To produce monoclonal antibodies, antibody producing cells (lymphocytes)can be harvested from an immunized animal and fused with myeloma cellsby standard somatic cell fusion procedures thus immortalizing thesecells and yielding hybridoma cells. Such techniques are well known inthe art. Hybridoma cells can be screened immunochemically for productionof antibodies which are specifically reactive with the oligopeptide, andmonoclonal antibodies isolated.

Polynucleotides and Expression

The proteins and fragments thereof described herein can be made byrecombinant processes and, therefore, may include amino acid sequencesderived from more than one species (i.e. chimeric constructs) or may beengineered to have a human, or human-like, amino acid composition (i.e.,a humanized construct). Accordingly, provided herein are vectorscomprising polynucleotides capable of encoding the described proteinsand fragments thereof. The vectors can be expression vectors.Recombinant expression vectors containing a sequence encoding apolypeptide of interest are thus provided. The expression vector maycontain one or more additional sequences such as, but not limited to,regulatory sequences (e.g., promoter, enhancer), a selection marker, anda polyadenylation signal. Vectors for transforming a wide variety ofhost cells are well known to those of skill in the art. They include,but are not limited to, plasmids, phagemids, cosmids, baculoviruses,bacmids, bacterial artificial chromosomes (BACs), yeast artificialchromosomes (YACs), as well as other bacterial, yeast and viral vectors.The vectors described herein may be integrated into the host genome ormaintained independently in the cell or nucleus.

A “vector” is a replicon, such as plasmid, phage, cosmid, or virus inwhich another nucleic acid segment may be operably inserted so as tobring about the replication or expression of the segment.

The term “operably linked” or “operably inserted” means that theregulatory sequences necessary for expression of the coding sequence areplaced in a nucleic acid molecule in the appropriate positions relativeto the coding sequence so as to enable expression of the codingsequence. By way of example, a promoter is operably linked with a codingsequence when the promoter is capable of controlling the transcriptionor expression of that coding sequence. Coding sequences can be operablylinked to promoters or regulatory sequences in a sense or antisenseorientation. The term “operably linked” is sometimes applied to thearrangement of other transcription control elements (e.g., enhancers) inan expression vector.

The terms “express” and “produce” are used synonymously herein, andrefer to the biosynthesis of a gene product. These terms encompass thetranscription of a gene into RNA. These terms also encompass translationof RNA into one or more polypeptides, and further encompass allnaturally occurring post-transcriptional and post-translationalmodifications. The expression/production of an antibody orantigen-binding fragment can be within the cytoplasm of the cell, and/orinto the extracellular milieu such as the growth medium of a cellculture.

Recombinant expression vectors contemplated to be within the scope ofthe description include synthetic, genomic, or cDNA-derived nucleic acidfragments that encode at least one recombinant protein which may beoperably linked to suitable regulatory elements. Such regulatoryelements may include a transcriptional promoter, sequences encodingsuitable mRNA ribosomal binding sites, and sequences that control thetermination of transcription and translation. Expression vectors,especially mammalian expression vectors, may also include one or morenontranscribed elements such as an origin of replication, a suitablepromoter and enhancer linked to the gene to be expressed, other 5′ or 3′flanking nontranscribed sequences, 5′ or 3′ nontranslated sequences(such as necessary ribosome binding sites), a polyadenylation site,splice donor and acceptor sites, or transcriptional terminationsequences. An origin of replication that confers the ability toreplicate in a host may also be incorporated. Such vectors may beintegrated into the host genome or maintained independently in the cellor nucleus.

The vectors described herein can be used to transform various cells withthe genes encoding the disclosed proteins. For example, the vectors maybe used to generate scaffold or antigen-binding protein-producing cellsor cell lines. Thus, another aspect features host cells transformed withvectors comprising a nucleic acid sequence encoding a protein. The hostcells disclosed herein can be prokaryotic or eukaryotic cells, Forexample the host cell can be a bacteria. In a preferred embodiment, thebacterial host cell is E. coli. Of course, the host cell can also be amammalian cell, such as a Chinese hamster ovary (CHO) cell line.Numerous other such host cells, prokaryotic and eukaryotic, are known inthe art and are considered to be within the scope of this disclosure.

Numerous techniques are known in the art for the introduction of foreigngenes into cells and may be used to construct the recombinant cells forpurposes of carrying out the inventive methods and producing proteins asdescribed herein, in accordance with the various embodiments describedand exemplified herein. The technique used should provide for the stabletransfer of the heterologous gene sequence to the host cell, such thatthe heterologous gene sequence is heritable and expressible by the cellprogeny, and so that the necessary development and physiologicalfunctions of the recipient cells are not disrupted. Techniques which maybe used include but are not limited to chromosome transfer (e.g., cellfusion, chromosome mediated gene transfer, micro cell mediated genetransfer), physical methods (e.g., transfection, spheroplast fusion,microinjection, electroporation, liposome carrier), viral vectortransfer (e.g., recombinant DNA viruses, recombinant RNA viruses) andthe like. Calcium phosphate precipitation and polyethylene glycol(PEG)-induced fusion of bacterial protoplasts with mammalian cells canalso be used to transform cells.

It is fully contemplated that the vectors such as those described hereincan be used to transform prokaryotic and/or eukaryotic cells tofacilitate expression of the described proteins. In some embodiments thedescribed vectors are used to facilitate protein expression in bacteria,such as E. coli. While any E. coli strain can be used to express theproteins described herein, some preferred strains include: BL21 (DE3),BL21-CodonPlus® (DE3)-RP, BL21-Codon Plus® (DE3)-RIL, BL21-(DE3)-pLysS(Stratagene). Eukaryotic cells can also be used with vectors tofacilitate protein expression. While those of skill in the art willrecognize that a wide variety of eukaryotic cells will be suitable forthis purpose, some preferred embodiments include mammalian cells andinsect cells. For example, in one embodiment Chinese hamster ovary (CHO)cells can be used with the vectors to facilitate expression of theprotein constructs provided herein. In alternative embodiments, insectcells, such as SD cells or S2 cells, can be used to with the describedvectors to facilitate expression of the protein constructs providedherein. Furthermore, those of skill in the art will understand thatvectors, not expressly disclosed herein, can be used for the samepurpose of expressing, or replicating nucleic acids encoding, thedescribed antigen binding proteins.

The described proteins can be encoded by a variety of polynucleotidescapable of encoding the amino acid sequences provided herein. Thesepolynucleotides can also be incorporated into vectors useful for themaintenance, replication, and/or expression of the polynucleotidesencoding the described antigen-binding proteins or the describedportions thereof. The vectors described above can be used to engineercells to express the antigen-binding proteins or the described portionsthereof encoded by the polynucleotides disclosed herein.

Compositions

Also described herein are compositions containing a protein or proteinsof the invention and a pharmaceutically acceptable carrier. Suchcompositions can be used to administer the described proteins to asubject or store or to maintain the described proteins. Any of thedescribed proteins can be used to produce such compositions, which mayinclude more than one of the disclosed proteins. In addition, suchcompositions can include other agents, such as therapeutic agents,preservatives, antimicrobial agents, and the like.

Described herein are compositions comprising at least one disclosedprotein and a pharmaceutically acceptable carrier. The compositions canbe formulated as any of various preparations that are known and suitablein the art, including those described and exemplified herein. In someembodiments, the compositions are aqueous formulations. Aqueoussolutions can be prepared by admixing the antigen-binding proteins inwater or suitable physiologic buffer, and optionally adding suitablecolorants, flavors, preservatives, stabilizing and thickening agents andthe like as desired. Aqueous suspensions can also be made by dispersingthe antigen-binding proteins in Water or physiologic buffer with viscousmaterial, such as natural or Synthetic gums, resins, methylcellulose,sodium carboxymethylcellulose, and other well-known suspending agents.

Also included are liquid formulations and solid form preparations whichare intended to be converted, shortly before use, to liquidpreparations. Such liquids include solutions, suspensions, syrups,slurries, and emulsions. Liquid preparations may be prepared byconventional means with pharmaceutically acceptable additives such assuspending agents (e.g., sorbitol syrup, cellulose derivatives orhydrogenated edible fats or oils); emulsifying agents (e.g., lecithin oracacia); non-aqueous vehicles (e.g., almond oil, oily esters, orfractionated vegetable oils); and preservatives (e.g., methyl orpropyl-p-hydroxybenzoates or sorbic acid). These preparations maycontain, in addition to the active agent, colorants, flavors,stabilizers, buffers, artificial and natural sweeteners, dispersants,thickeners, solubilizing agents, and the like. The compositions may bein powder or lyophilized form for constitution With a suitable vehiclesuch as sterile water, physiological buffer, saline solution, oralcohol, before use.

The compositions can be formulated for injection into a subject. Forinjection, the compositions described can be formulated in aqueoussolutions such as water or alcohol, or in physiologically compatiblebuffers such as Hanks's solution, Ringer's solution, or physiologicalsaline buffer. The solution may contain formulatory agents such assuspending, stabilizing and/or dispersing agents. Injection formulationsmay also be prepared as solid form preparations which are intended to beconverted, shortly before use, to liquid form preparations suitable forinjection, for example, by constitution with a suitable vehicle, such assterile water, saline solution, or alcohol, before use.

The compositions can be formulated in sustained release vehicles ordepot preparations. Such long acting formulations may be administered byimplantation (for example subcutaneously or intramuscularly) or byintramuscular injection. Thus, for example, the compositions may beformulated with suitable polymeric or hydrophobic materials (forexample, as an emulsion in an acceptable oil) or ion exchange resins, oras sparingly soluble derivatives, for example, as a sparingly solublesalt. Liposomes and emulsions are well-known examples of deliveryvehicles suitable for use as carriers for hydrophobic drugs.

The proteins described herein may be administered orally in anyacceptable dosage form such as capsules, tablets, aqueous suspensions,solutions or the like. The proteins may also be administeredparenterally including but not limited to: subcutaneous, intravenous,intramuscular, intra-articular, intra-synovial, intrasternal,intranasal, topically, intrathecal, intrahepatic, intralesional, andintracranial injection or infusion techniques. Generally, the proteinswill be intravenously or intraperitoneally, for example, by injection.

The subject can be any animal, and preferably is a mammal such as amouse, rat, hamster, guinea pig, rabbit, cat, dog, monkey, donkey, cow,horse, pig, and the like. In some embodiments, the mammal is a human. Insome embodiments, the mammal is other than a human.

All publications and other references mentioned herein are incorporatedby reference in their entirety, as if each individual publication orreference were specifically and individually indicated to beincorporated by reference. Publications and references cited herein arenot admitted to be prior art.

The present invention is not intended to be limited by any theory. Thisinvention will be better understood by reference to the ExperimentalDetails which follow, but those skilled in the art will readilyappreciate that the specific experiments detailed are only illustrativeof the invention as defined in the claims which follow thereafter.

Experimental Details

Examples are provided below to facilitate a more complete understandingof the invention. The following examples illustrate the exemplary modesof making and practicing the invention. However, the scope of theinvention is not limited to specific embodiments disclosed in theseExamples, which are for purposes of illustration only.

Example 1

Heparin-induced thrombocytopenia (HIT) is an autoimmune thromboticdisorder caused by immune complexes containing platelet factor 4 (PF4),antibodies to PF4, and heparin or cellular glycosaminoglycans (GAGS). Wesolved the crystal structures of the: 1) PF4 tetramer/fondaparinuxcomplex, 2) PF4 tetramer/KKOFab complex (a murine monoclonal HIT-likeantibody), and 3) PF4 monomer/RTO-Fab complex (a non-HIT anti-PF4monoclonal antibody). Fondaparinux binds to the ‘closed’ end of the PF4tetramer and stabilizes its conformation. This interaction in turnstabilizes the epitope for KKO on the “open” end of the tetramer.Fondaparinux and KKO thereby collaborate to “stabilize” the ternarypathogenic immune complex. Binding of RTO to PF4 monomers prevents PF4tetramerization and inhibits KKO and human HIT IgG-induced plateletactivation and platelet aggregation in vitro, and thrombus progressionin vivo. The atomic structures provide a basis to develop newdiagnostics and non-anticoagulant therapeutics for HIT.

Introduction

Heparin-induced thrombocytopenia/thrombosis (HIT) is a potentially fatalimmune complex mediated thrombotic disorder that develops inapproximately 1% of patients exposed to unfractionated heparin (UFH) or,less commonly, to low-molecular-weight heparins (LMWH) (Linkins 2012).HIT is caused by antibodies to complexes that form between plateletfactor 4 (PF4) released from activated platelets and heparin or cellularglycosaminoglycans (GAGS). Circulating immune complexes composed of PF4,heparin, and HIT antibodies bind to platelet and monocyte Fcy receptorsand promote cellular activation, leading to generation of thrombin(Phillips 2008; Lee 2013; Lewis 2003). Anti-PF4/heparin antibodiesdetected by ELISA also develop in a high proportion of patients exposedto heparin, e.g., after cardiopulmonary bypass surgery, even in theabsence of clinical complications. However, the basis for thedistinction between pathogenic and non-pathogenic antibodies is unknown,which can lead to over-diagnosis and overtreatment (Cuker 2012).

In prior studies, we identified a murine monoclonal antibody (KKO) toPF4/heparin complexes that causes heparin-induced thrombosis andthrombocytopenia in a murine model, thus sharing salient features withthe clinical disorder (Arepally 2000). Human HIT antibodies compete withKKO for binding to PF4/heparin, and KKO augments formation of pathogenicimmune complexes (Sachais 2012). An isotype matched anti-PF4 antibody(RTO) that binds comparably to PF4, but does not cause pathogeniccomplexes (Zhang 1994), has also been identified.

Here, we describe and compare the crystal structures of PF4 in complexwith Fabs derived from KKO and RTO and the structure of PF4 in complexwith a heparin-mimic pentasaccharide, fondaparinux. These resultssuggest that by stabilizing the structure of the asymmetric PF4tetramer, fondaparinux might foster the binding of HIT antibody andformation of the pathogenic ternary complex. Surprisingly, the non-HITantibody RTO binds to an epitope expressed on the surface of the PF4monomer that partly overlaps with the KKO epitope in the tetramer.Binding of RTO prevents tetramerization of PF4 and inhibits KKO-inducedplatelet activation and aggregation in vitro and has potent inhibitoryeffects on thrombus progression in vivo.

These crystallographic data lead to a model to help understand thestructural basis of the pathogenic immune complex that causes HIT at theatomic level and provide a structural basis for the development of newdiagnostics and non-anticoagulant therapeutics for HIT. The datadescribed herein also provides insights into the process through which anormal human protein becomes “antigenic” to the mammalian immune systemafter complexing with endogenous or exogenous GAGS.

Results Crystal Structure of the PF4/Fondaparinux Complex: Generation ofthe HIT Antigen

Based on previously reported crystal structures, human PF4 can assume anasymmetric tetrameric configuration. Each monomer containsthree-stranded antiparallel β-sheets upon which an aperiodic N-terminaldomain and an amphipathic C-terminal α-helix are folded 9. NMR studiesreveal that PF4 exists as an equilibrium among monomers, dimers andtetramers (Mayo 1989). The tetrameric structure of PF4 is stabilized notonly by salt bridge interactions between dimers AB/CD (hereafter we usethe nomenclature for the monomer chains of PF4 as in PDB: 1RHP 9), butalso by the antiparallel β-sheet-like structures in the N-termini ofdimers AC or BD 9. It had been assumed that the asymmetry of the PF4tetramer plays a role in heparin binding (Mayo 1995) and several modelshave been proposed to explain the interaction between heparin and PF4(Cowan 1986; Stuckey 1992; Nguyen 2015). However, conclusive structuraldata at the atomic level have not been reported.

Binding of pathogenic HIT antibodies to PF4 is markedly enhanced byheparin or various glycosaminoglycans (GAGS), which are themselveshighly heterogeneous in size and composition and unsuitable forcrystallization. To investigate how the GAG might induce or augmentneoantigen formation, we crystallized PF4 in complex with fondaparinux,a homogenous synthetic pentasaccharide heparin that forms complexes withPF4 as assessed by atomic force microscopy and photon correlationspectroscopy (Greinacher 2006). Fondaparinux induces anti-PF4/heparinantibodies as do other GAGS (Bauer 2001; Turpie 2002; Warkentin 2005)and can occasionally cause HIT (Rota 2008; Warkentin 2008; Warkentin2007; Bhatt 2013).

We solved the crystal structure of PF4 in complex with fondaparinux as amodel GAG. The PF4/fondaparinux complex is a pseudo-222 tetramer formedby a pseudo 2-fold non-crystallographic symmetry (NCS) and a 2-foldcrystallographic symmetry perpendicular (FIG. 9A, Table 1 forstatistics). Fondaparinux appears as a well-defined molecule, evidencedby the strong and clear electron densities (FIG. 1A and FIG. 1B).Superposition of the bound and non-bound structures of PF4 tetramer(PDB:1RHP) using Ca gives a root-mean-square deviation of only 0.76 Å.This indicates that the association with fondaparinux causes minimalconformational changes in PF4 except for slight re-direction of sidechains around the binding groove, which is consistent with CD studiesreported recently (Brandt 2014; Kreimann 2014).

TABLE 1 Summary of Crystalloaraphic data and Refinement StatisticsKKOFab/PF4 RTOFab/PF4 Data Collection PF4/Fondaparinux KKOFab tetramermonomer Beamline X4A/X4C(BNL) X6A(BNL) X4A/X4C(BNL) X4A/X4C(BNL)wavelength (Å) 0.9791 0.9795 0.9792 2.07 Space group P3₂21 P2₁2₁2₁P2₁2₁2₁ P2₁2₁2 Cell dimensions a = 67.33 Å a = 41.48 Å a = 49.49 Å a =161.42 Å b = 67.33 Å b = 92.12 Å b = 99.34 Å b = 171.87 Å c = 61.76 Å c= 122.14 Å c = 261.74 Å c = 208.16 Å α = β = 90°, γ = 120° α = β = γ =90° α = β = γ = 90° α = β = γ = 90° Resolution * (Å)   2.50 (2.54-2.50)  2.20 (2.28-2.20)   4.10 (4.17-4.10)   3.75 (3.81-3.75) Rsym or Rmerge0.057 (0.72)  0.088 (0.66)  0.089 (0.44)  0.154 (1.65)  I/σI 22.6 (3.0) 10.8 (2.2)  7.9 (1.9) 6.8 (2.9) Completeness (%) 98.2 (98.6) 94.4 (98.5)87.9 (61.3) 99.4 (98.9) Redundancy 10.3 (10.0) 5.1 (4.9) 8.9 (4.5) 26.5(25.6) Refinement Resolution (Å) 22.76-2.50 30.8-2.20 20-4.11 50-3.74No.unique reflections 5913 24599 10642 60619 R_(work)/R_(free)0.224/0.245 0.200/0.262 0.317/0.382 0.257/0.297 No. atoms Protein 9733322 8548 30074 Solvent 17 99 0 0 B-factors (Å²) Protein 95 37 268 134ligand 157 NA NA NA R.m.s deviations Bond lengths (Å) 0.006 0.008 0.0060.007 Bond angles (°) 0.93 1.17 0.92 1.34 Ramachandran (%)Favored/disallowed 100/0  99.4/0.3  97.1/0.8  97.4/1.0  * Values in theoutermost shell are given in parentheses

The PF4 tetramer displays a pseudosymmetry characterized by an ‘open’end and a ‘closed’ end that can be defined by the intra-chain distancesbetween amino acids E28 and K50. E28 and K50 are approximately 3 Å apartand form stable salt bridge pairs between chain A and chain C. However,the same inter4 chain salt bridge pairs do not form between E28 and K50in chains B and D, which are about 8 Å apart (FIG. 2 ). This asymmetryin the PF4 tetramer explains why calculation of its electrostaticpotential identifies only two positively charged grooves on the ‘closed’side of PF4 tetramer surface to which fondaparinux can bind (FIG. 9D).

PF4 tetramers can be formed between the AB and CD dimers, or between theAC and BD dimers (Zhang 1994). Fondaparinux binds among monomers A, Band C or monomers A, C, and D (FIG. 9B). This interaction stabilizes theAB/CD association and the AC/BD association, thus further stabilizingthe PF4 tetramer. We suggest that this stabilization might represent avery early step in the pathogenesis of HIT antigen.

Analysis of the crystal lattice further reveals that fondaparinux notonly makes contact with a single PF4 tetramer within the groove formedby three monomers (ABC or ACD), but also at a second site involving theC-terminal helix of the third monomer (B or D). It has been proposedthat the HIT antigen develops when charge neutralization by polyanionsallows positively charged PF4 tetramers to undergo close approximation(Nguyen 2015; Greinacher 2006). The crystal structure of thePF4/fondaparinux complex provides a more complete atomic understandingof this process. On fondaparinux molecule not only binds within thegroove on the surface of one PF4 tetramer, it is also shared with asecond symmetry-related tetramer through binding to its C-terminal helix(FIG. 9C). Most of these interactions are between the sulfate groups offondaparinux and the basic residues in PF4. The finding that twotetramers of PF4 bind to one heparin fragment provides insight into theinitiation of ultralarge complex formation.

Based on what is observed in the crystal, we propose the followingpathway for the formation of the heparin/PF4 complex. Heparin firstbinds to the groove in one PF4 tetramer. Binding of the first PF4tetramer imparts a local linearized structure on heparin. This enhancesthe binding of a second tetramer. Progression of this process eventuatesin the formation of the large antigenic complex in which PF4 tetramerscluster around a semi-rigid linear heparin chain (FIG. 9F). Clusteringmight be required for apposition of sufficient HIT antibodies to inducepersistent activation of cellular FcγIIA receptors (Reilly 2001). Thismodel provides structural insight into a recently proposed heparin/PF4interaction model (Nguyen 2015), and is also consistent with previousNMR (Mayo 1995) and site-directed mutagenesis studies (Ziporen 1998; Li2002). Moreover, this set of atomic structures extends other studiesshowing a heparin chain of about 10 saccharides is required to form astable antigenic complex (Nguyen 2015; Kreimann 2014; Visentin 2001;Greinacher 1995).

Crystal Structure of PF4/KKO-Fab: Recognition of the “Open” End of PF4Tetramer Leads to a Structural Model of the HIT Pathogenic TernaryComplex.

To understand the structural basis for the formation of pathogenicimmune complexes, we next solved the crystal structure of human PF4complexed with the Fab fragment of the monoclonal antibody KKO (FIG.10A, Table 1 for statistics). KKO recapitulates the salient features ofHIT in a mouse model in vivo and competes with human platelet activatinganti-PF4/heparin antibodies in vitro (Cuker 2013; Reilly 2001). TheKKO-Fab fragment binds to the PF4 tetramer by making contacts with threemonomers (for example, chains A, B and D within one PF4 tetramer, FIG.10A, surface representations; FIG. 10B, detail of KKO epitope on the PF4tetramer surface). This structure provides strong support for theconcept that formation of the tetramer is required for optimal bindingof KKO (Amiral 1995).

KKO does not bind to murine (m) PF4 complexed with heparin. Therefore,we compared the sequence of human (h) PF4 with mPF4, and made structurebased mutants based on the proposed contact sites identified in theco-crystal (FIG. 3 ). Mutations in the putative KKO epitope essentiallyabolished binding of the HIT antibody KKO to these variant PF4s by ELISA(FIG. 10C) and did not support heparin/KKO-induced platelet activationas measured by light transmission aggregometry in contrast to wild-typePF4 (Arepally 2000).

Further analysis revealed that PF4 within the PF4/KKO-Fab complex adoptsa symmetric tetrameric structure, which is an ‘open’-‘open’, possiblyintermediate, conformation (FIG. 4 ). This result enabled us tosuperimpose the PF4/KKO Fab complex onto the PF4/heparin complex model(FIG. 9F) to build a structural model of the KKO-Fab/PF4/heparin ternarycomplex. In this model, the polysaccharide binds to the ‘closed’ end ofPF4, stabilizing the PF4 tetramer, which orients the ‘open’ end forrecognition by KKO. In this way, fondaparinux and HIT-like antibodycollaborate to “stabilize” the ternary complex (FIG. 10E).

These structures provide the first atomic level description of thepathological complex that might occur in HIT. The model might also helpto explain why fondaparinux, a short fragment of heparin, which isantigenic, only rarely causes HIT. Although the protein-GAG interactionscan in theory be propagated to additional PF4 tetramers and fondaparinuxmolecules, we propose that covalent bonding between pentasaccharideunits in longer heparins markedly increases the stability of theholo-complex. Consequently, the more “stable” complex becomes moreantigenic by enhancing antibody avidity.

Crystal Structure of PF4/RTO-Fab: A Non-Pathogenic Antibody Recognizesthe PF4 Monomer

RTO is an anti-PF4 antibody that possesses the same isotype as KKO, butdoes not cause HIT in vivo (Arepally 2000; Rauova 2006). In contrast toKKO, binding of RTO to hPF4 is not enhanced by heparin (Arepally 2000).RTO and KKO also do not compete for binding to hPF4 by ELISA (Sachais2012). However, KKO and RTO display the same Bmax when measured byELISA, and is thus illustrative of the difficulty in distinguishinghuman pathogenic and non-pathogenic antibodies using contemporary bulkequilibrium assays (Sachais 2012). The reason why RTO does not cause HITwas revealed through atomic analyses.

To help understand the difference between the impacts of these twoantibodies, we next solved the crystal structure of the RTO-Fab incomplex with hPF4 (FIG. 11A, Table 1 for statistics). Unexpectedly,RTO-Fab binds only to the PF4 monomer. This was affirmed by a change inthe migration of PF4 monomer, but not PF4 tetramer, upon addition ofRTO-Fab assessed by FPLC (FIG. 5 ).

Also unexpectedly, the RTO epitope on PF4 overlapped considerably withthe KKO epitope (compare FIG. 11B with FIG. 10B, FIG. 6 ). In thestructure of the RTO-Fab/PF4 complex, the C-terminal helices still packagainst the β-sheet domain. However, the orientation of the C-terminalhelices shifts approximately 60 degrees relative to the β-sheet coredomain (FIG. 11C). Superimposition of the PF4 asymmetric tetramer ontothe RTO-Fab/PF4 complex reveals that RTO binds between monomer A andmonomer B and prevents the formation of the AB dimer, and therebyprevents formation of the tetramer (formation of an AC dimer is stilltheoretically possible) (FIG. 11D). Together, these structural data leadus to propose that tetramerization of PF4, which is required to form theHIT antigen, would be disrupted by the non-HIT antibody RTO.

RTO Impedes PF4 from Forming Tetramers and Thereby Prevents KKO InducedPlatelet Activation: Implications for Clinical Management.

Based on the knowledge that PF4 tetramers exist in equilibrium withmonomers and dimers, and with even higher ordered complexes when heparinis present, we predicted that RTO might prevent or disrupt tetramerformation and thereby prevent KKO-induced platelet activation in thepresence of heparin. Indeed, RTO inhibited the activation of plateletsby KKO and human HIT IgG in the presence of heparin in a dose-dependentmanner, as assessed by expression of P-selectin on flow cytometry (FIG.12A). Preincubation of PF4 with RTO also inhibited KKO-induced plateletaggregation (FIG. 12B).

Moreover, RTO completely inhibited KKO-induced progression of thrombosisin a laser microvascular injury model in mouse PF4−/−/human PF4+/+/humanFcRγIIA+ mice 26 (FIG. 12C and FIG. 12D). These data indicate that thenon-HIT antibody RTO, when engineered and humanized, could provide atemplate for the development of non-anticoagulant intervention in HIT.

Discussion

It is uncertain how heparin or an endogenous glycosaminoglycan convertsa normal host protein, PF4, into an “autoantigen”. The crystallographicdata provide insight into the multistep process of neoantigen formationat the atomic level. We report the first crystal structure of the PF4monomer, here in complex with the Fab fragment of an anti-PF4 antibodyRTO, which affirms that PF4 exists in solution as an equilibrium amongmonomers, dimers, and tetramers (Mayo 1989). In the monomeric state, theN-terminal amino acids of PF4 adopt a flexible extended conformation andthe C-terminal helices swing around atop the β-sheet core domain. Incontrast, in the tetrameric state the individual monomers undergo amarked conformational change. The C-terminal helices of the PF4 monomersare all re-orientated and re-aligned and the N-termini of the monomersform antiparallel β-sheet-like structures. The repulsive forces betweenthe positively charged C-terminal helices of monomer A and monomer B inthe AB dimer (and similarly between monomer C and monomer D in the CDdimer) might induce thermal instability in the tetramer (Nesmelova2008). Heparin binds among PF4 monomers and also bridges PF4 tetramers,which helps overcome this instability and leads to the formation ofstable individual tetramers, a fundamental first step in the expressionof the neoantigen in HIT.

The data described herein also indicates that the asymmetry of thefondaparinux-bound PF4 tetramer, which has an “open” end and a “closed”end, plays an important role in forming the pathogenic antigeniccomplex. Based on these findings, we propose that heparin or a GAGfragment stabilizes the PF4 tetramer by clamping the monomers togetherthrough the “closed” end, orienting the “open” end in the asymmetric PF4tetramer that comprises the binding site for KKO. Thus, in the absenceof heparin, the KKO binding site on the tetramer is nonexistent orincompletely expressed on the PF4 monomer and dimer and is onlytransiently expressed and unstable on tetramers that form at highconcentrations of PF4. Thus, stabilization of the tetramer by heparinleading to a more stable orientation of an epitope on each PF4 tetramer,compared with the transient and more random orientation of the epitopein the absence of heparin, is a second defining step in the process ofgenerating the HIT neoantigen. Heparins or GAGs of sufficient lengthscan then cluster several PF4 tetramers further enhancing stability,which helps to explain why PF4, among heparin binding proteins, isespecially antigenic (Sachais 2012; Nguyen 2015). In the third step,HIT-like antibody KKO recognizes the stabilized “open” end of the PF4tetramers, further promoting formation of the pathogenic ternarycomplex.

PF4 species are known to participate in coagulation, cell growth andangiogenesis (Lippi 2010; Funatsu 2001; Maione 1990).

PF4 monomers can associate into a tetrameric form as well as dissociatefrom that tetrameric species. The PF4 tetramer that interacts withheparin a highly sulfated glycosaminoglycan, or oligonucleotidesfacilitate the expression of an antigenic epitope that can bindantibodies to form a larger complex.

The ability of monomeric PF4 to form stable tetramers can be affected bydeletion or amino acid substitutions. In addition while monomericmutants may still bind heparin, they are not able to form canonicalclusters with heparin as wildtype pf4 does.

PF4 which has been altered and can no longer form stable tetramerscannot form antigenic complexes when exposed to heparin, and will not beable to express the complete antigenic epitope that binds pathogenicantibodies.

The studies described herein were performed with fondaparinux becausethe heterogeneity of clinical heparins and GAGs preclude obtainingstructural detail. Therefore, we do not exclude the possibility that lowmolecular weight or unfractionated heparin might induce additionalchanges within and between PF4 tetramers (Brandt 2014; Block 2014).Indeed, the structural model described herein predicts that when a GAGlonger than about 10 saccharides binds to more than one PF4 tetramer,additional structural changes within the tetramers themselves are quitelikely. These structural changes are more likely to occur within theinner surface of the PF4 tetramers in the cluster along the PF4/heparinbinding interface than primarily at the HIT antibody binding epitope,which lies on the outer surface. Therefore, additional studies areneeded based on this model, to determine if these additional structuralchanges in PF4 imparted by longer GAGs directly modulate the contactsites between antibody and antigen.

The A32-A39 loop on the PF4 monomer appears to be highly immunogenic.However, the data described herein suggest that pathogenic andnon-pathogenic antibodies that bind to this region differ in how theyaffect the monomer-dimer tetramer equilibrium. Pathogenic antibodiesthat act like KKO and preferentially recognize the tetrameric speciesdisplay greater avidity in the presence of heparin, which approximatesepitopes, and in turn the bound antibody fosters oligomerization(Sachais 2012). Binding of KKO may also shift the equilibrium fromheparin wrapping around the PF4 species suggested in a previous model 13by forcing it into a more stable linear conformation (Nguyen 2015). Thenet result would be the generation of stable ultralarge immune complexes(Rauova 2005) capable of sustained activation of FcγIIA receptors onplatelets and monocytes (Reilly 2001; Rauova 2010). In contrast,anti-PF4 antibodies that preferentially recognize monomers in the mannerof RTO are readily detected by contemporary ELISAs, but they do notgenerate large immune complexes that cause disease. Indeed, anti-PF4antibodies that act like RTO may actually compete with pathogenicantibodies by preventing or disrupting tetramer formation and therebylimit formation of larger immune complexes as depicted in FIG. 5 . Thesedata provide a new mechanistic model for the development of a humanautoimmune disease, in which a host-protein PF4 complexed with host GAGsassumes diverse oligomeric conformations that differentially bindautoreactive antibodies leading to diverse clinical outcomes.

The studies described herein might have implications for the diagnosisof HIT. The two prototypic antibodies we studied, RTO and KKO, bindcomparably to PF4/heparin on ELISA plates at equilibrium and do notcompete with each other for binding in this format (Sachai 2012; Cuker2013). This suggests that ELISA wells might contain PF4 in diverseconformations ranging from monomer through tetramer and likely higherordered complexes. Studies are in progress to determine if ELISA formatsbased on more homogenous populations of PF4 complexes and/or mutant PF4monomers will reduce detection of non-pathogenic, potentially“blocking”, anti-PF4 antibodies. Additional studies will also be neededto determine if differences in the ratio of pathogenic and “blocking”antibodies might contribute to the likelihood of developing HIT.

The studies described herein might also have implications for therapy.Anticoagulants are the standard treatment for HIT, but their efficacy isincomplete and dosing is limited by the risk of bleeding (Cuker 2012;Kang 2015) Our data indicate that RTO binds to PF4 monomers and preemptsassembly of stable tetramers and, as a result, inhibits KKO (and humanHIT IgG) induced platelet activation and aggregation in vitro and,importantly, progression of antibody-induced thrombosis in vivo. It islikely release of PF4 and formation of antigenic complexes extendsbeyond diagnosis and introduction of a direct thrombin inhibitor, asdoes the risk of recurrent thromboembolic complications. The inhibitoryeffects of RTO indicate that tetramerization of PF4 is targetable andthat this antibody may provide a structural basis for developingrational non-anticoagulant HIT-specific intervention for this seriousand common iatrogenic disorder.

It has been difficult to differentiate between the impact of changeswithin the secondary structure of individual PF4 tetramers that affectantibody affinity and effects on tetramer oligomerization that enhanceavidity, because both contribute to antibody binding measured in ELISAwells that likely contain various conformations of PF4. Our use of Fabfragments that are unable (or show little ability) to oligomerize PF4tetramers, and fondaparinux, which is antigenic but rarely pathogenicbecause it has a low capacity to form higher ordered structures,provided an opportunity to delineate the evolution of the antigenic sitewithin the PF4 tetramer itself.

Nevertheless, we wish to emphasize that our studies also have potentiallimitations. First, the necessity of using fondaparinux as the GAGprecluded possible additional relevant changes within PF4 induced bylonger heparin molecules that are more likely to induce HIT. Nor can weassess the intramolecular and intermolecular effects on PF4 imparted bythe complex array of cellular GAGs. Second, although most HIT antibodiescompete with KKO for binding, our data do not preclude additionalcontact sites between polyspecific human antibodies and PF4/heparin.Third, crystallography and other biophysical approaches using purifiedproteins inherently explore atomic interactions in a single stablestructure favored by the experimental conditions. Thus, the full rangeof dynamic changes in (super) oligomerization that occur during theevolution of the disease may not be captured. Thus, our study onlyprovides a model that may help to explain sentinel events through whichGAGs may induce binding of autoantibodies to a normal host protein.Fourth, in our experiments, RTO was added prior to KKO. The dynamics ofRTO binding to PF4 might well be different when heparin and a pathogenicHIT antibody are already present. We are currently investigating theeffect of RTO on thrombus growth in vivo following exposure to KKO.

Methods Expression and Purification of Human PF4 and Antibodies.

Wild type (WT) hPF4 and hPF4 mutants in plasmid pMT/BiP/V5-His(Invitrogen Corp., Carlsbad Calif.), were expressed using the DrosophilaExpression System (Invitrogen), purified, and characterized as described(Sachais 2012). Briefly, the protein was collected in serum-free mediumInsect-Xpress (Lonza, Walkersville, Md.) and isolated by affinitychromatography using a HiTrap Heparin HP column (GE Healthcare) on anAKTA Purifier (GE Healthcare) at 4° C. and eluted at 1.8 M NaCl (wtPF4)using a linear gradient. Fractions containing purified PF4 detected bysilver staining of 12% polyacrylamide gels (SDS-PAGE) were pooled,concentrated and buffer exchanged into 50 mM HEPES, 0.5 M NaCl, pH ˜7.2using an Amicon Ultra filter (3000 molecular weight cut-off, Millipore).Protein was quantified using a BCA assay (Pierce). To obtain the PF4mutants, PCR with corresponding primers (Table 2) was performed onpMT/BiP/V5/HisA-uPA plasmid under conditions recommended by TheQuikChange Site-Directed Mutagenesis Kit manual (Stratagene, La Jolla,Calif.). The resulting plasmids were sequenced to confirm the mutation.

TABLE 2 Sequences of primers that were used to generate PF4 mutants.Primers Sequences 9SCV GAA GAT GGC GAC CTG AGC TGC GTG TGT GTG AAG ACC(SEQ ID NO: 17) 9SCV-anti GGT CTT CAC ACA CAC GCA GCTCAG GTC GCC ATC TTC (SEQ ID NO: 18) 55R AGG AAA ATT TGC TTG GAC CGCCAA GCT CCG CTG TAC (SEQ ID NO: 19) 55R-anti GTA CAG CGG AGC TTG GCG GTCCAA GCA AAT TTT CCT (SEQ ID NO: 20)

The murine anti-human PF4 IgG2bK monoclonal antibodies KKO and RTO havepreviously been described (Arepally 2000). The IgGs were purified fromconditioned PFHM-II media (Invitrogen) using protein A-agarose(Invitrogen) as recommended by the manufacturer. IgG purity wasdemonstrated by SDS-PAGE on NuPAGE 4-12% Bis-Tris Gel (Invitrogen). Fabfragments were generated by papain digest using Pierce® Fab PreparationKit (Thermo Scientific, Rockford, Ill.) essentially as recommended bythe manufacturer, followed by three rounds of removing of Fc fragmentswith protein A agarose beads, and extra purification with anti-mouse IgG(Fc-specific) (Sigma M4280) and anti-mouse IgG (Fab-specific) SigmaM4155 antibodies bound to CNBr-activated Sepharose 4 Fast Flow beads(Amersham Biosciences Corp., Piscataway, N.J.) as recommended by themanufacturer. KKOFab, RTOFab and PF4 were further purified bysize-exclusion column on an AKTA purifier system (GE Healthcare). HumanHIT IgG was purified using staph protein agarose (source) from apheresate obtained from a patient with HIT.

ELISA Assays.

Binding of human IgG was measured essentially as previously describedfor KKO and RTO antibodies (Sachais 2012). Briefly, Immulon 4 HBX96-well plates (Thermo Fisher Scientific, Waltham, Mass.) were coatedovernight with either PF4 or PF4 mutant at 5 ug/ml. The plates wereincubated for 30 min with either PBS (control) or with 0.5%glutaraldehyde at room temperature, extensively washed and blocked with1% BSA in PBS. The plates were incubated with human patient IgG samplesat experimentally selected concentration of 20 μg/ml for 30 min at 37°C. IgG 11 binding was measured as absorbance at 405 nm (A405) afterincubation for 30 min at 37° C. with horseradish peroxidase-conjugatedImmunoPure Goat Anti-Human IgG (H+L), HRP Conjugated Product No. 31412(Pierce. Rockford, Ill.) diluted 1:10,000 in 1% BSA/PBS. Horseradishperoxidase substrate ABTS was from Roche Applied Science, Penzberg,Germany. Absorbance was measured with a SpectraCount plate reader(Packard BioScience, Waltham, Mass.).

In Vitro Platelet Activation Mediated by KKO+PF4

Blood for in vitro studies (platelet activation and light transmissionaggregometry) was collected after informed consent from healthy,aspirin-free volunteers using a 19-gauge butterfly needle in 129 mMsodium citrate (10:1, vol/vol) under protocols approved by theInstitutional Review Board of the University of Pennsylvania and theChildren's Hospital of Philadelphia. Whole blood samples were incubatedin Ca++/Hepes buffer (2.5 mM CaCl2, 1.25 mM MgCl2, 150 mM NaCl, 10 mMHEPES, pH 7.5) 1/100 v/v in the presence of APC labeled antihCD41 and PElabeled anti-P-selectin, PF4 (10 μg/ml) and the concentrations of RTOMOAb indicated in the figure for 15 min at room temperature. KKO (20μg/ml) or human HIT IgG (500 μg/mL) was added for 20 min at roomtemperature, samples were then diluted by adding 400 μL of HBSA/BSA/EDTAbuffer and immediately measured by flow cytometry (BD LSRFortessa™).Platelets were gated based on the forward-scatter and CD41 fluorescenceparameters, and binding of anti-P-selectin antibodies was quantified asgeometric mean fluorescent intensity (MFI).

Light Transmission Aggregometry

Blood was centrifuged for 12 min at 210×g at 25° C. to generateplatelet-rich plasma (PRP) and at 900×g for 10 min at 25° C. to produceplatelet-poor plasma (PPP). Platelet aggregation was measured in PRPusing a dual-channel lumiaggregometer (model 700, Chrono-logCorporation, Havertown, Pa.) per the manufacturer's instructions. Allexperiments were completed within 4 hrs of blood collection. PRP (500μl) was pre-warmed for 2 min at 37° C., heparin (Sagent Pharmaceuticals,Schaumburg, Ill.) was added (final concentration 0.1 U/ml) for 30 secfollowed by wild type or mutant rPF4 (final concentration 10 μg/ml) for30 sec followed by KKO or RTO (100 μ/ml) for up to 10 min. The finalvolume of added reagents did not exceed 5% of the starting volume ofPRP. To examine inhibition of KKO induced platelet aggregation, 100 μgRTO was preincubated with 5 μg PF4 for 15 min at 25° C. Heparin (finalconcentration 0.1 U/ml) was added to pre-warmed PRP. Thirty secondslater PF4 or PF4/RTO complex was added (final concentrations in PRP 10μg/ml and 10 μg/ml/200 μg/ml, respectively). KKO (final concentration of100 μg/ml) was added 30 sec later and aggregation was assessed as above.

Inhibition of KKO-Induced Thrombosis In Vivo.

Transgenic male C57BL mice, lacking mouse PF4 but expressing human PF4and human FcγRIIA (Reilly 2001) were studied. Mice were matchedlittermates between 6-10 weeks of age. The cremaster laser injury model(Rauova 2006; Falati 2002) was used to visualize in vivo thrombusformation. After surgical preparation, Alexa 647 (BD Biosciences)labeled mouse CD41-F(ab′)2 fragments were infused intravenously to labelcirculating platelets. Each mouse then received 50 μg/g of either RTO orthe IgGK2B isotype control TRA intravenously followed by 7-8 focalarterial injuries. A brightfield and fluorescence snapshot of eachinjury was taken 15 minutes after the initial injury. KKO was theninjected intravenously at a dose of 2.5 μg/g. Fifteen minutes afterinjection of KKO, a second brightfield and fluorescence snapshot of thesame injuries was taken to compare platelet deposition before and afterKKO. Image analysis was performed using Slidebook 6 (31 IntelligentImaging Innovations, Denver, Colo.). The size of the thrombus wasdetermined by having the software automatically segment the image usingOtsu thresholding to remove background and then calculate and export thesize of the platelet fluorescence in microns (Phillips 2008). Theinvestigator was not blinded during these studies. All experiments wereperformed in compliance with the institutional guidelines for the careand handling of experimental animals were approved by the Children'sHospital of Philadelphia Institutional Animal Care and Use Committee.

Statistical Analysis

Statistical analysis of in vivo injuries was performed using Prism 6(GraphPad, La Jolla, Calif.). A two-tailed Student's t-test undernon-parametric conditions with Mann Whitney correction was performed toassess statistical significance. P values <0.05 were consideredsignificant. There was no randomization of mice. The mice used wereselected based on availability. There was no exclusion criteriaestablished prior to experimentation, available healthy mice wereselected for use. No tests of normality or power analyses wereconducted.

Crystallization and Data Collection

Purified KKOFab (5 mg/ml) and RTOFab (5 mg/ml) were mixed with hPF4 atdifferent ratios and incubated on ice overnight before setting upcrystallization trials. The hPF4/fondaparinux complex was prepared byadding 6-fold molar excess fondaparinux (Arixtra, Sanofi-Synthelabo LLC)into purified hPF4 (7 mg/ml); the mixture was incubated on ice in abuffer containing PBS and 0.3 M NaCl.

Crystallizations of hPF4/fondaparinux, KKOFab, hPF4/KKOFab, andhPF4/RTOFab were performed using the hanging-drop vapor diffusion methodby mixing the protein and well solution at 1:1 volume ratio at 16° C.Crystallization kits from Hampton Research and Molecular Dimensions wereused for initial crystallization trials. Optimized hPF4/fondaparinuxcomplex crystals were obtained in the well solution containing 2%PEG4000, 17% MPD, 0.1 M sodium acetate pH 5.6. hPF4/fondaparinux complexcrystals were then directly flashcooled in liquid nitrogen by using theMiTeGen micromounts (MiTeGen, LLC).

Diffraction quality KKOFab crystals were obtained in the well solutioncontaining 14% PEG2000, 0.06 M zinc acetate, 0.1 M sodium cacodylate,pH6.8. KKOFab crystals were transferred into the well solutionsupplemented with 25% glycerol, soaked for one second, and thenflash-cooled in liquid nitrogen. Diffraction quality hPF4/KKOFabcrystals were obtained in the well solution containing 7% PEG6000, 0.1 MTris-HCl, pH 7.8. hPF4/KKOFab complex crystals were flashcooled inliquid nitrogen similarly as KKOFab crystals. hPF4/RTOFab complexcrystals were obtained in the well solution containing 0.2 M ammoniumsulfate, 0.1 M BIS-TRIS, pH 6.5, 25% w/v Polyethylene glycol 3350. Thediffraction quality PF4/RTOFab crystals were optimized by macro-seeding.hPF4/RTOFab complex crystals were transferred into the well solutionsupplemented with 20% glycerol, soaked for one second, and thenflash-cooled in liquid nitrogen.

All crystallographic data sets were collected at 100 K with ADSC CCDdetectors. The long-wavelength (A-2.07 Å) sulfur anomalous diffractiondata sets for hPF4/fondaparinux complex and hPF4/RTOFab complex werecollected at beamline X4A at National Synchrotron Light Source (NSLS) atBrookhaven National Laboratory (Upton, N.Y. USA). Other data sets werecollected at NSLS beamlines X6A and X4C at Brookhaven NationalLaboratory. All diffraction data were processed by using the HKL-2000package (Otwinowski 1997).

Structure Determination and Refinement.

All structures were solved by molecular replacement. The structure ofthe hPF4/fondaparinux complex was solved by CCP4 program MOLREP (Winn2011; Vagin 2010) with AB dimer from the hPF4 structure (PDB IDcode:1F9Q) as a search model. The structure of KKOFab was also solved byMOLREP with the Fab structure from the DsbB-Fab complex (PDB IDcode:2ZUQ) as a search model. The structure of the hPF4/KKOFab complexwas solved by CCP4 program Phaser (McCoy 2007) with the refined KKOFabstructure and hPF4 (PDB ID code: 1RHP) structure as search models. Thestructure of the hPF4/RTOFab complex was solved by Phaser using thestructure of 2H2 Fab fragment of immature Dengue virus (PDB IDcode:4KVC) and the structure of the A chain of the hPF4 monomer (PDB IDcode: 1F9Q) as search models. The final solution has 8 hPF4/RTOFabcomplexes, related by noncrystallographic symmetry (NC S), in anasymmetric unit.

All models were iteratively built in COOT (Emsley 2010) and refined byREFMAC (Murshudov 1997; Murshudov 2011) or PHENIX (Afonine 2012).Refinement of the hPF4/KKOFab complex at low resolution was performedusing deformable elastic network (DEN)-assisted refinement (Schroder2010), REFMAC and OPUS-XREF (Lu 2006). To assist model building, the lowresolution electron density maps for the hPF4/KKOFab complex wereoptimized using Bfactor sharpening. The anomalous diffraction datacollected at long wavelength were used to assist model building andrefinement of the hPF4/fondaparinux and hPF4/RTOFab complexes. Thequality of refined models was checked by program PROCHECK (Laskowski1993) and MOLPOBITY (Chen 2010). All structural figures were prepared inPyMol (The PyMOL Molecular Graphics System, Schrödinger, LLC., availableon the world wide web at pymol.org). The electrostatics potentials werecalculated by program APBS, an adaptive Poisson-Boltzmann solver (Baker2001; M 2006). Data collection and refinement statistics are listed inTable 1.

Acknowledgement

We thank scientists at National Synchrotron Light Source X4A, X4C andX6A at the Brookhaven National Laboratory for their assistance in datacollection. We thank Drs. Bruce S. Sachais, Ann H. Rux, Rustem I.Litvinov and John W. Weisel for advice and discussions. This work wassupported in part by NIH grant P01HL110860 (DBC).

Accession Numbers

Atomic coordinates and structure factor files have been deposited in theProtein Data Bank (PDB) under the accession codes 4R9W forhPF4/pentasaccharide complex, 4R97 for KKOFab, 4R9Y for hPF4/KKOFabcomplex, 4RAU for hPF4/RTOFab complex.

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DISCUSSION

Heparin-induced thrombocytopenia (HIT) is thrombotic disorder caused byimmune complexes containing antibodies to an antigen composed ofplatelet factor 4 (PF4) and heparin or cellular glycosaminoglycans(GAGS). The structure of these immune complexes and how theircomposition might contribute to the difference between pathogenic andnon-pathogenic anti-PF4 antibodies are unknown. To address thesequestions, the inventors solved the crystal structures of humanrecombinant PF4 in complex with Fabs derived from KKO (a murinemonoclonal HIT-like antibody that competes with pathogenic human HITantibodies) and RTO (an isotype-matched non-HIT anti-PF4 antibody)combined with the crystal structure of PF4 complexed with theheparin-mimic pentasaccharide fondaparinux as a model sugar. The PF4tetramer is asymmetric and is capable of accommodating only twofondaparinux molecules. Fondaparinux binds between monomers A, B and Cor between monomers A, C, and D, which stabilizes the AB/CD and AC/BDassociations and the resultant tetramer. KKO-Fab binds to the PF4tetramer by making contacts with now identified residues within each ofthree PF4 monomers, indicating that tetramerization of PF4 is a criticalinitiating step in antigen formation. Mutations in the putative KKOepitopes in PF4 abolished antibody binding. Unexpectedly, RTO-Fab bindsto the PF4 monomer between the AB dimer interface. Importantly, theamino acid sequence recognized by RTO and KKO show considerable overlap.However, the epitope for RTO is obscured upon tetramer formation, indirect contrast to binding of KKO, which requires tetramer formation tobind. Binding of RTO to the PF4 monomer prevents formation of AB dimersand subsequent tetramerization. In support of these findings,preincubation of PF4 with RTO inhibits KKO induced platelet activationand platelet aggregation in vitro. Without wishing to be bound by anyscientific theory, based on the analyses of crystal lattices, thepresent disclosure proposes a new model of the heparin/PF4 complex, inwhich PF4 tetramers cluster around a semi-rigid linear heparin subunit.Clustering of PF4 on heparin might be required for apposition ofsufficient HIT antibodies to induce persistent activation of cellularFcγIIA receptors. Heparin and pathogenic HIT antibodies collaborate tostabilize the ternary immune complex, which leads to the disappearanceof binding sites for at least some non-pathogenic HIT antibodies. Thebalance between anti-monomer and anti-tetramer PF4 antibodies may helpdetermine the probability of clinical disease. This model also helps toexplain why RTO-like anti-PF4 antibodies are found so commonly inasymptomatic patients exposed to heparin and why fondaparinux may beantigenic but rarely causes HIT, whereas longer heparin fragments andGAGS extend and render the holo-complex more stable and thereby fosterthe formation of pathogenic immune complexes. In summary, withoutwishing to be bound by any scientific theory, these crystallographicstudies lead to a new model to explain the formation of pathogenicimmune complexes that lead to HIT. The inhibitory effect of the anti-PF4antibody RTO provides a structural basis for the development of newdiagnostics and non-anticoagulant therapeutics.

PF4 monomers can associate into a tetrameric form as well as dissociatefrom that tetrameric species. The PF4 tetramer that interacts withheparin a highly sulfated glycosaminoglycan, or oligonucleotidesfacilitate the expression of an antigenic epitope that can bindantibodies to form a larger complex.

The ability of monomeric PF4 to form stable tetramers can be affected bydeletion or amino acid substitutions. In addition while monomericmutants may still bind heparin, they are not able to form canonicalclusters with heparin as wildtype pf4 does.

PF4 which has been altered and can no longer form stable tetramerscannot form antigenic complexes when exposed to heparin, and will not beable to express the complete antigenic epitope that binds pathogenicantibodies.

Example 1 hereinabove describe and compare the crystal structures of PF4with Fabs derived from KKO and RTO and the structure of a heparin-mimicpentasaccharide in complex with human PF4. Without wishing to be boundby any scientific theory, these structural studies reveal that thetetramerization of the antigen PF4 induced by heparin is a criticalinitiating step in the pathogenesis of HIT. KKO recognizes tetramericPF4 and stabilizes the pathogenic ternary complex with heparin.Surprisingly, the non-HIT antibody RTO binds to an epitope that overlapswith the KKO epitope on the surface of PF4, prevents PF4 from beingtetramerized, and inhibits KKO induced platelet activation in vitro andthrombocytopenia in vivo.

The information presented herein indicates a potential alternativeapproach to diagnosing and treating HIT. Example 1 described hereinaboveprovides the first crystal structures of PF4 in a complex with aspecific part RTO or KKO (the fragment antigen binding region). Thesestructures give novel insight into how each antibody interacts with PF4,and this knowledge indicates a new approach for diagnosing and treatingHIT. For example, the present invention provides methods andcompositions for the treatment of HIT based on the non-pathogenicantibody, which blocks the action of the pathogenic antibody and limitsthe development of HIT. The present invention also provides riskassessment and diagnostic tests for HIT.

The knowledge of KKO and RTO presented herein is useful in generatingantibody assays to diagnose and/or monitor the progression of HIT asdescribed herein. Furthermore, insight into the non-pathogenic RTOantibody (specifically, how it prevents KKO-induced HIT) is useful forgenerating antibody-based therapies for HIT.

1-48. (canceled)
 49. A method for treating a subject afflicted withheparin-induced thrombocytopenia (HIT) comprising administering to thesubject an effective amount of a mutant platelet factor 4 (PF4) monomercomprising amino acids 9 to 70 of SEQ ID NO: 11 and having a serine atamino acid position 9 relative to the amino acid sequence of SEQ ID NO:11.
 50. the method of claim 49, wherein the mutant PF4 monomer furthercomprises a valine at amino acid position 11 relative to the amino acidsequence of SEQ ID NO:
 11. 51. The method claim 49, wherein theN-terminal amino acid residue of the mutant PF4 monomer is amino acid atposition 9 of SEQ ID NO:
 15. 52. The method of claim 49, wherein theadministering of the mutant PF4 monomer interferes with PF4 tetrameroligomerization.
 53. The method of claim 49, wherein the mutant PF4monomer further comprises glutamic acid at amino acid position 50relative to SEQ ID NO:
 11. 54. The method of claim 49, wherein themutant PF4 monomer further comprises arginine at amino acid position 55relative to the amino acid sequence of SEQ ID NO:
 11. 55. The method ofclaim 49, wherein the mutant PF4 monomer further comprises glutamic acidat amino acid position 50 and arginine at amino acid position 55relative to SEQ ID NO:
 11. 56. A method for treating a subject afflictedwith heparin-induced thrombocytopenia (HIT) comprising administering tothe subject an effective amount of a mutant platelet factor 4 (PF4)monomer consisting of amino acids 9 to 70 of SEQ ID NO: 11 and having aserine at amino acid position 9 and a valine at amino acid position 11relative to the amino acid sequence of SEQ ID NO:
 11. 57. The method ofclaim 56, wherein the administering of the mutant PF4 monomer interfereswith PF4 tetramer oligomerization.
 58. A method for treating a subjectafflicted with heparin-induced thrombocytopenia (HIT) comprisingadministering to the subject an effective amount of a mutant plateletfactor 4 (PF4) monomer comprising amino acids 9 through 70 of SEQ ID NO:11, a K50E substitution relative to the amino acid sequence of SEQ IDNO: 11, and at least one amino acid mutation selected from Q9S, L11V andL55R relative to the amino acid sequence of SEQ ID NO:
 11. 59. Themethod of claim 58, wherein the administering of the mutant PF4 monomerinterferes with PF4 tetramer oligomerization.
 60. The method of claim58, wherein the N-terminal amino acid residue of the mutant PF4 monomeris amino acid at position 9 of SEQ ID NO:
 15. 61. The method of claim58, wherein the mutant PF4 monomer consists of amino acids 9 through 70of SEQ ID NO: 11 having Q9S and K50E substitutions relative to the aminoacid sequence of SEQ ID NO: 11, and at least one amino acid mutationselected from L11V and L55R relative to the amino acid sequence of SEQID NO:
 11. 62. The method of claim 61, wherein the administering themutant PF4 monomer interferes with PF4 tetramer oligomerization.